Calcium and Sperm

Physiol Rev 91: 13051355, 2011
doi:10.1152/physrev.00028.2010
CALCIUM CHANNELS IN THE DEVELOPMENT,

MATURATION, AND FUNCTION OF SPERMATOZOA
Alberto Darszon, Takuya Nishigaki, Carmen Beltran, and Claudia L. Trevio
Departamento de Gentica del Desarrollo y Fisiologa Molecular, Instituto de Biotecnologa, Universidad Nacional
Autnoma de Mxico, Cuernavaca, Morelos, Mxico
Darszon A, Nishigaki T, Beltran C, Trevio CL. Calcium Channels in the Development,
L
Maturation, and Function of Spermatozoa. Physiol Rev 91: 13051355, 2011;
doi:10.1152/physrev.00028.2010.A proper dialogue between spermatozoa and
the egg is essential for conception of a new individual in sexually reproducing animals.
Ca2 is crucial in orchestrating this unique event leading to a new life. No wonder that
nature has devised different Ca2-permeable channels and located them at distinct sites in
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 1, 2017

spermatozoa so that they can help fertilize the egg. New tools to study sperm ionic currents, and
image intracellular Ca2 with better spatial and temporal resolution even in swimming spermato-
zoa, are revealing how sperm ion channels participate in fertilization. This review critically examines
the involvement of Ca2 channels in multiple signaling processes needed for spermatozoa to
mature, travel towards the egg, and fertilize it. Remarkably, these tiny specialized cells can express
exclusive channels like CatSper for Ca2 and SLO3 for K, which are attractive targets for
contraception and for the discovery of novel signaling complexes. Learning more about fertilization
is a matter of capital importance; societies face growing pressure to counteract rising male
infertility rates, provide safe male gamete-based contraceptives, and preserve biodiversity through
improved captive breeding and assisted conception initiatives.
I. INTRODUCTION 1305 has been long recognized (51, 79). More recently, the mo-
II. MAMMALIAN SPERMATOGENESIS 1307 lecular cloning of the calcium sensing receptor (CaSR) es-
III. CALCIUM CHANNELS IN... 1309 tablished its role as a first messenger (61). To function in
IV. MAMMALIAN SPERM MATURATION 1317 these roles, Ca2 concentration must be finely regulated
V. MAMMALIAN ACROSOME REACTION 1328 both intra- and extracellularly. The intracellular Ca2 con-
VI. SPERM CHEMOTAXIS IN... 1333 centration ([Ca2]i) is maintained within strict limits by a
VII. SEA URCHIN ACROSOME REACTION 1337 variety of cellular mechanisms that buffer, sequester, ex-
VIII. CONCLUDING REMARKS 1341 trude, and accumulate Ca2, with concentration changes
often occurring in highly localized areas within the cell (79).
Indeed, Ca2 plays a pivotal role in fertilization participat-
I. INTRODUCTION
ing in the main functions of both invertebrate and verte-
brate spermatozoa such as maturation, motility, and the
Fertilization is a fundamental and convoluted process, nec-
acrosome reaction (AR), an exocytotic process required for
essary to unite two haploid gametes, the male spermato-
fertilization in many species (571). The fundamental impor-
zoon and the female oocyte/egg, to produce a unique indi-
tance of ion channels, particularly those for Ca2, for sperm
vidual. Mature and competent gametes are needed to
achieve this impeccably choreographed process. We are still physiology and fertilization has been consolidated in the
far from fully understanding the gamete intracellular mo- last 10 years due to significant advances in the tools avail-
lecular dialogue necessary for fertilization. Spermatozoa able to study them (135, 292, 519). This is emphasized by
are specialized cells able to reach, recognize, and fuse to the the fact that certain Ca2 channel blockers inhibit these
egg or oocyte despite being essentially incapable of gene functions (139, 178, 381, 419).
transcription or protein synthesis. Improving our under-
standing of fertilization is essential as it is at the heart of Cells use a significant portion of their energetic resources to
creating life. Furthermore, it is urgently needed to tackle build and maintain ion concentration gradients across their
increasing male infertility in developed societies, to provide membranes using ion pumps and transporters. These ionic
safer male gamete-based contraceptives, to improve animal gradients are a source of information about the state of
breeding, and to preserve biodiversity (37, 521). the cell and its surroundings. Ion channels, which upon
activation can alter the electric potential of the cell and
Ca2 is critical in different cellular signaling processes act- the concentrations of internal second messengers within
ing at diverse spatial sites, and its role as second messenger a wide time-range, depending on the modes of channel
1305
CALCIUM CHANNELS AND SPERMATOZOA
regulation, translate this information into cell behavior describe and discuss the Ca2 channels that participate in
to contend with the changes within and outside of the these key sperm processes. It is organized considering the
cell. Therefore, it is not surprising that genomes from the development and travel of spermatozoa towards the egg.
fruit fly to the human encode hundreds of different chan- In general, the characteristics of each channel family ap-
nels. pear when first involved in a process or function. There-
after, each new section provides further information re-
Spermatozoa are small, morphologically complex cells (FIG. 1) garding properties of specific channels participating in a
and differentiated (FIG. 2) to accomplish a concrete but particular sperm function.
fundamental goal: to deliver their genetic material to the
egg, activate its development, and generate a new individ- Ion channels in general constitute a very minor percentage
ual. To achieve this goal, a matter of life and death, they of the total membrane protein. In this regard, demonstrat-
must contend with myriad environmental changes while ing their functional presence and location in a specific cell is
they mature and then steer in search of the egg (FIG. 3). In an arduous and sometimes frustrating enterprise. Because
this regard, it would seem that the multiple signaling spermatozoa are basically considered transcriptionally si-
processes needed for spermatozoa to mature, properly lent, their ion channels are synthesized by spermatogenic
swim, and reach the egg at the right time, in a proper cells, their progenitors, during spermatogenesis. However,

condition and fuse with it, would require a rich set of ion the presence of transcripts of a specific ion channel, and
channels and transporters. This review will attempt to even of its protein and currents in spermatogenic cells, does
FIGURE 1. Diagram of sea urchin, mouse, and human spermatozoa. The major sperm components for each
species are indicated. A: sea urchin sperm; entire cell body (left), enlarged head cross-section (top right)
showing the position of the acrosome, nucleus, centriole, and nuclear envelope remnants (above and below the
nucleus). Note a single doughnutlike mitochondrion that resides below the nucleus. Enlarged cross-section of
flagellum (bottom right) shows the classic structure of 92 doublets microtubules, in which the nine doublets
surround a central pair. Outer and inner dynein arms as well as the radial spokes are illustrated. The flagellum
of this species does not have additional cytoskeleton around the axoneme. B: entire cell bodies of mouse and
human spermatozoa. The shape of the head and the acrosome are quite different in these species. Mouse
spermatozoa have a hook and crescent moon-shaped head and acrosome, respectively. In contrast, human
spermatozoa have a rounded and cap-shaped head and acrosome, respectively. Mammalian flagella can be
divided into mid, principal, and end pieces. The midpiece is separated from the principal piece by the annulus.
Equatorial and postacrosomal segments are also indicated. In mouse, a cytoplasmic droplet is usually observed
in testicular and epididymal spermatozoa, but not in ejaculated mature spermatozoa, while it is frequently
observed in human mature spermatozoa. C: enlarged human sperm (head and midpiece region) cross-sections
viewed from two different angles (90 rotation). The nucleus occupies most of the head space, and it is overlaid
by the acrosome; there is very little cytosol in these cells. The centriole and the redundant nuclear envelope are
located at the base of the head. The axoneme is surrounded by outer dense fibers in the midpiece and the
principal piece. The outer dense fibers are surrounded by mitochondria in the midpiece, but by the fibrous
sheath in the principal piece. D: enlarged cross-section of mammalian sperm flagellum. The axoneme runs
along all the flagella; it is surrounded by mitochondria and outer dense fibers in the midpiece, by the outer
dense fibers, and the fibrous sheath in the principal piece (2 out of 9 outer dense fibers are fused with the
fibrous sheath). The end piece contains only the axoneme.
1306 Physiol Rev VOL 91 OCTOBER 2011 www.prv.org

DARSZON ET AL.

FIGURE 2. Diagram of the mammalian male reproductive organs and spermatogenesis. Left: spermatogen-
esis diagram. Spermatogenesis advances from the base to the lumen of the seminiferous tubule with
structurally and functionally essential nurse cells, called Sertoli cells. Spermatogonia (germinal stem cells)
reside in the basal compartment and proliferate. Primary spermatocytes (leptotene) differentiate from sper-
matogonia and transverse the blood-testis barrier formed by tight junction of adjacent Sertoli cells. Secondary
spermatocytes (pachytene and diplotene) are formed by the first meiosis and converted into round spermatids
by the second meiosis. Elongated spermatids are cells in the process of spermiation undergoing the remark-
able morphological transformation of the nucleus and cell body. In the final process of spermiation, most of the
cytoplasm will be removed (phagocytized by Sertoli cells) to form fully developed spermatozoa (the residual
cytoplasm is known as cytoplasmic droplet). Middle: a seminiferous tubule and its cross-section. Newly formed
spermatozoa are transported towards the epididymis through the lumen of the tubule (L). Right: testis and
epididymis. The testis is filled with tightly packed seminiferous tubules; one of them is represented as a gray line
inside the testis. The epididymis consists of a single highly coiled tubules. This organ can be roughly divided into
three portions: caput, corpus and cauda; sperm transit along these tubules is required for sperm maturation.
not necessarily mean that the protein will be functionally II. MAMMALIAN SPERMATOGENESIS
expressed in mature spermatozoa. The physiological rele-
vance of the transcripts detected in mature spermatozoa is Spermatogenesis is the transformation of diploid spermato-
still unclear and cannot be taken as proof that the proteins gonial stem cells into haploid spermatozoa that takes place
they code for are present in spermatozoa. Therefore, dem- inside the seminiferous tubules (FIG. 2). It is a continuous,
onstrating that an ion channel is functionally present in complex, and highly regulated process. Functional units
mature spermatozoa requires proving either by controlled along the tubules epithelial compartments (basal and adlumi-
immunological or proteomic strategies that the protein is nal) repeat a cycle that can be divided in four main phases:
there, and showing that it is functional either electrophysi- mitosis, meiosis, spermiogenesis, and spermiation. In the
ologically or using ion-sensitive dyes or proteins. Pharma- basal compartment, primary spermatogonia proliferate and
cology and ultimately elimination of the specific ion channel after a species-specific fixed number of mitotic divisions
from spermatozoa, in the species where it can be done (i.e., (253) differentiate into leptotene spermatocytes that are the
null mice), will yield clear evidence of the role of the channel only cell type that transverses the blood-testis barrier into
in sperm physiology. the adluminal compartment. Once in the adluminal com-
partment, cells differentiate to pachytene and then diplo-
The authors are aware of their limitations and preferences, tene spermatocytes that undergo two rounds of meiotic di-
which most likely have led to some biased proposals and vision to produce haploid spermatids. The process contin-
regrettably to exclude some important contributions. Sev- ues with spermiogenesis in which spermatids experience a
eral excellent reviews are provided, hoping they will com- strong morphological transformation including, among
plement and balance the subject matter of this review (47, others, the formation of the acrosome, chromatin conden-
178, 418, 433, 548). sation, and the elimination of the excess cytoplasm (residual
Physiol Rev VOL 91 OCTOBER 2011 www.prv.org 1307


FIGURE 3. Diagram of the mammalian female reproductive system. Left: the architecture and major
components of the female reproductive tract are shown. The vagina is the site of ejaculation and the start point
of the sperm path towards the oocyte. Swimming and muscle contractions allow spermatozoa to pass through
the cervix and the uterus to reach the isthmus. The cervix separates the vagina from the uterus, which is the
site of fetus development. The isthmus or sperm reservoir is located in the Fallopian tubes. In the absence of
oocytes, spermatozoa remain attached to the epithelium that lines the tubal lumen for several days, depending
on the species. The ampulla is the second segment of the Fallopian tube and the site of fertilization. During
ovulation, the oocyte migrates to the ampulla, and spermatozoa detach from the oviductal epithelium (sperm
can attach and detach several times) to reach the egg. Middle: cross-section of the isthmus and ampulla
regions showing the vast invaginations of these segments. Right: diagram of the oocyte and the surrounding
zona pellucida and cumulus cells.
body) with the simultaneous formation of the cytoplasmic inside the tubules and hormonal stimulation is provided by
droplet (see below), to produce elongated spermatids that Leydig cells from the interstitial compartment where peri-
migrate towards the edge of the lumen (118). During sper- tubular myoid cells are also present (212). The germ cell
miation, fully developed spermatozoa are released into the cycle and movement along the tubule is a process under
lumen. Two interesting structures are formed in the final tight control including signaling that involves several fami-
stages of sperm differentiation. During nuclear volume re- lies of kinases and phosphatases (246). In spite of the im-
duction, the excess material is packaged into the redundant portance of spermatogenesis, the regulation as well as bio-
nuclear envelope (RNE), whose name derives from the lack chemical and molecular mechanisms underlying this pro-
of an established function for this organelle (FIG. 1). This cess have remained largely unexplored (118). In particular,
structure has been detected in spermatozoa from several very little is known about the role of Ca2 during the cel-
mammals and in sea urchin (210), and it is now proposed lular events required to produce mature spermatozoa. Con-
that it functions as a Ca2 store. In support of this proposal, sidering that Ca2 is one of the most common messengers
inositol trisphosphate (IP3) receptors (IP3Rs) have been de- inside the cell and has such a predominant role in almost
tected in the RNE (261). The second structure found in every signaling cascade, it is likely that it would be involved
mammalian sperm is the cytoplasmic droplet (see FIG. 1) in this process. Indeed, spermatogenic cells regulate their
that is produced during the formation of the residual body Ca2 homeostasis with a fine balance between Ca2 uptake
which concentrates excess cytoplasm, mitochondria, lipid and release by intracellular stores (250). In fact, postmeiotic
droplets, the protein synthesis machinery, residual struc- cells (round spermatids) and meiotic cells (pachytene sper-
tures, etc. The residual body is detached from sperm during matocytes) differentially regulate their [Ca2]i upon exter-
spermiogenesis, and it is phagocytosed by Sertoli cells, but nal supply of oxidative glycolytic substrates such as glucose
the cytoplasmic droplet remains attached to the flagella, and lactate (434). For example, exposure of mouse sper-
and depending on the species, this droplet is lost in the matogenic cells to maitotoxin (a potent marine toxin) pro-
epididymis or after ejaculation (247). There are several produced a significantly greater elevation in [Ca2]i in cells
posed functions for the cytoplasmic droplet, such as the incubated in glucose-containing media than in cells incu-
modification of the sperm plasma membrane (394) or vol- bated in lactose-containing media (435). Glucose and lac-
ume regulation (173). tate are normally secreted by Sertoli cells into the adluminal
compartment. It was proposed that these differences in
Spermatogenic cells do not accomplish this process inde- Ca2 signaling, mediated primarily by internal Ca2 stores,
pendently; instead, they are nursed by Sertoli cells (FIG. 2) could be a differentiation-related process (434). Intracellu-

DARSZON ET AL.
lar Ca2 channels (see sect. IIID), namely, IP3Rs (378) and ogy and electrophysiological approaches (11, 14, 234, 324,
ryanodine receptors (RyRs), have been detected in sper- 451). The main interest was as a proxy to understand their
matogenic cells, and for the latter, a role during differenti- role in mature sperm, but their participation during differ-
ation has been proposed (119). entiation has not been investigated. In particular, currents
from voltage-dependent Ca2 (CaV) channels were detected
To ensure a high-quality supply of gametes, spermatogenic and characterized in mouse spermatogenic cells (134, 234,
cells undergo apoptosis, a form of programmed cell death, 479, 514), and their function was extrapolated to mature
to discard excess and unfit cells (518); however, the signal- sperm.
ing mechanism leading to apoptosis is not completely un-
derstood. Apoptosis can also be induced by environmental Although spermatozoa are in principle transcriptionally si-
factors such as temperature, toxins, exposure to chemicals, lent, as mentioned, certain mRNAs have been detected in
etc. Herrera et al. (250) reported that high temperature these cells. It has been proposed that these RNAs are long
conditions can induce programmed cell death in rat sper- lived and can be delivered to the egg for later expression
matogenic cells mediated through a Ca2-initiated mecha- (174, 300). A few papers have reported that some sperm
nism. Mishra et al. (363) treated rat spermatogenic cells in mRNAs can be translated to protein via the mitochondria
vitro with 2,5-hexanedione (a common industrial solvent) machinery (231, 232). In any case, mRNAs from either

to induce apoptosis. They characterized an apoptotic mech- spermatogenic cells or mature sperm coding for several CaV
anism regulated by changes in the [Ca2]i that translate into -subunits and auxiliary subunits have been isolated, and
a differential expression of members of the Bcl protein fam- the presence of the corresponding proteins has been con-
ily. These proteins can be either pro- or antiapoptotic fac- firmed with specific antibodies (see sect. IIIA and TABLE 1).
tors, and their expression ratio determines the survival or
death of the cells (363). In accordance with the observed
III. CALCIUM CHANNELS IN
pharmacology, Mishra et al. (363) proposed that the chan-
SPERMATOZOA
nels involved belong to the CaV3 family (see sect. IIIA).
As mentioned before, very little is known about Ca2 reg- A. Voltage-Gated Ca2 Channels
ulation and its contribution during spermatogenesis. Be-
cause mature spermatozoa are basically transcriptionally Voltage-gated Ca2 (CaV) channels are responsible for
silent and spermatozoa were not easily patch clamped (162) [Ca2]i increases induced by membrane potential (Em)
until very recently (292), spermatogenic cells were the pre- changes that regulate processes such as contraction, secre-
ferred model to study Ca2 channels using molecular biol- tion, neurotransmission, and gene expression in diverse cell
Table 1. Expression of Cav channels in sperm cells

Channel mRNA WB ICC Detectable Mice KO Reference Nos.
Cav1.1 Die at birth 288

Cav1.2 Mo Hu Su Mo Su Mo (h,pp) Hu (mp) Su (f) Lethal 164, 219, 223, 288,
400, 514, 556
Cav1.3 fertile 413
Cav1.4 Retinal defects 342
Cav2.1 Mo Mo Mo (h,f) Die at 1 288, 324, 556
month
Cav2.2 Hu Mo Mo (h,pp) Spermatogenic Fertile 272, 400, 554
Cav2.3 Mo Hu Su Mo Su Mo (h,pp) Hu (pp,h) Su (h, f) Spermatogenic Fertile 223, 324, 514, 556
Cav3.1 Mo Hu Mo (h,f) Hu (mp,h) Fertile 134, 164, 400, 479, 514
Cav3.2 Mo Hu Mo (h) Hu (f) Spermatogenic Fertile 114, 134, 164, 400,
Testicular sperm 478, 514
Cav3.3 Mo Mo (mp) Hu (mp) NA 134, 400, 514
Catsper 1 Mo Hu Mo Mo (pp) Epididymal sperm Infertile 292, 432
Catsper 2 Mo Hu Mo Mo (pp) Infertile 425
Catsper 3 Mo Hu Mo Mo (f,h) Infertile 278, 335, 423
Catsper 4 Mo Hu Mo Mo (f,h) Infertile 278, 335, 423
The detection of the mRNA, the protein by Western blot (WB), or the subcellular localization (h, head; f,
flagella; pp, principal piece; mp, midpiece) by immunocytochemistry (ICC) for each CaV subunit and different
species is shown. The detection of the specific current (if present) and the knockout (KO) phenotype (Mo,
mouse; Hu, human; Su, sea urchin) is indicated. NA, not available.

types. These channels have been catalogued into two major ylglycerol (DAG), and PKC; and 3) a mechanism involving
functional classes: high voltage-activated (HVA) and low Gs that does not involve any kinases.
voltage-activated (LVA) channels (89). HVA channels re-
quire strong depolarizations to open, after which they inac- Ca2 itself regulates CaV channel inactivation by associat-
tivate slowly. Considering the biophysical and pharmaco- ing with CaM. The mechanisms involved in this regulation
logical characteristics of their macroscopic currents, they are best understood for CaV1 and CaV2 channels, although
have been classified into L, N, P/Q, and R types. On the CaV3 channel function may also be influenced by CaM
other hand, LVA channels need weaker depolarizations to (336). Other related Ca2 sensor proteins resembling CaM
open, and they inactivate faster and at more negative po- can displace it from shared binding sites in the 1 subunits
tentials than HVA channels. LVA currents were initially of CaV channels, but are different enough to confer distinct
named T type (181) because they open transiently. forms of regulation (89, 149, 310).
The ion-conducting pore of CaV channels is formed by the Only recently has the modulation of CaV3 channels by en-
1 subunit of 200 kDa, which is encoded by a family of 10 dogenous ligands and second messenger pathways been ex-
genes grouped into three subfamilies: CaV1 with four mem- plored (113). Molecules such as anandamide, arachidonic
bers CaV1.1-CaV1.4 all conducting L-type currents, CaV2 acid, and polyunsaturated fatty acids (micromolar range)

with three members conducting P/Q- (CaV2.1), N- inhibit CaV3 channels. For example, lipoaminoacids such
(CaV2.2), and R-type (CaV2.3) currents, and CaV3 with as N-arachidonoylglycine (NAGly), a molecule closely re-
three members (CaV3.1-CaV3.3) all conducting T-type cur- lated to endocannabinoids that does not activate cannabi-
rents. In this review, we use CaV1 and/or CaV2 for HVA noid receptors or TRPV1 channels, has been reported to
and CaV3 for LVA channels. block CaV3.2 channels (34).
The topology of the 1 subunit consists of four repeated Interestingly, Zn2, an important divalent cation present at
domains (IIV) each containing six transmembrane (TM) millimolar concentrations in seminal plasma (in mammals,
segments (S1S6) surrounding a central pore (87, 91).
from 0.2 to 2.6 mM depending on the species; Refs. 331,
Auxiliary subunits for CaV1 and CaV2 channels form
349), preferentially inhibits CaV3.2 (IC50 0.8 M), while
another protein family with four members (Ca V 2,
CaV3.1 and Cav3.3 are 100- to 200-fold less sensitive. Ap-
CaV, CaV, and CaV) (88). CaV channels in different
parently, some of the Zn2 inhibitory effects are mediated
tissues may display unique properties due to alternative
through binding to an extracellular histidine residue in the
splicing, posttranslational modifications (10), and di-
S3S4 linker of domain I (265).
verse regulation mechanisms. In what follows, the prin-
cipal regulation modes of CaV channels will be summa-
Truly selective CaV3 blockers are urgently needed to help char-
rized, paying attention to those more relevant to sperm
acterize these channels, both in heterologous expression and in
physiology. TABLE 2 sums up the pharmacology of CaV
native cells. Approaches aimed at developing specific blockers
channels.
such as the one undertaken by Uebele et al. (523) are welcomed.
Several protein kinases such as protein kinase A (PKA), They described a specific CaV3 antagonist: 2-(4-cyclopropylphe-
protein kinase C (PKC), and Ca2/calmodulin (CaM)- nyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]pyridin-2yl}ethyl)
dependent protein kinase II (CaMKII) phosphorylate and acetamide (TTA-A2) that is a selective and state-dependent
regulate CaV channels. In many instances, these phos- CaV3 channel antagonist, with a potency of 10 nM in the
phorylation events stimulate CaV channels by diverse depolarized (inactivated) state and 400 nM in the hyperpo-
mechanisms depending on the particular isoform (38, 87, larized (closed) state. Sensitivity of L-, N-, P/Q-, and R-type
89, 128, 551). currents to TTA-A2 was above 6 M. The authors used this
compound therapeutically to treat mice with obesity and
CaV channels are also regulated by G proteins activated sleep disorders that were correlated with CaV3.1 activity.
through different pathways including those activated by
neurotransmitters and hormones (140). This regulation can The regulatory properties of auxiliary subunits have been
be direct, via physical interactions between the G protein described mainly for CaV1 and CaV2 (HVA) channels.
and channel subunits or indirect via second messengers Although there is conflicting evidence showing that CaV1
and/or by protein kinases (140, 152). Recently, Perez-Reyes and CaV2 auxiliary subunits may or may not modulate
(405) published an excellent review on the properties of CaV3 channels, recent reports show that among the eight
CaV3 channels. This work describes three different mecha- members of the CaV auxiliary channel subunits, 6
nisms involving activation of G protein-coupled receptors inhibits CaV3.1 currents (115, 326) and that the amino
that lead to particular inhibition of CaV3.2 channels: 1) a acid sequence GxxxA (where x is any amino acid) in its
direct inhibition by G22 subunits; 2) a pathway indepen- first transmembrane domain plays an important role in
dent of G22 but involving phospholipase C (PLC), diac- this modulation. Chen and Best (115) proposed that the

DARSZON ET AL.
Table 2. Calcium channel blockers inhibit acrosomal reaction (AR)

Calcium Channel Concentration Used, in M
Blocker Target (IC50, M) Inducer (Species) Effect in AR (IC50 for AR) Reference Nos.
CaV blocker
Calciseptine Cav1 (0.43)# rZP3 (Hu) Inhibition 3 145,# 282

Diltiazem Cav1.2 (12 5)### rZP3 (Hu) Inhibition 30 282, 540###
Cav3.2 (96.2 37.8)## High pH/depolarization Inhibition (10) 176, 408##
or ZP3 (Bu,Ram)
Egg jelly (Su) Inhibition (55) 287
Nifedipine Cav1 (2.7)* ZP3 (Hu) Inhibition (60%) 1.2 120, 282, 579*
Cav3.1 (109)** ZP3 (Hu) Inhibition 100 120, 282,
464**
Cav3.2 (21.3 1.3)## ZP4 (Hu) Inhibition (1.6) 120, 408##
ZP3 (Mo) Inhibition 10 391

ZP (Hu) No effect 20 52
Progesterone (Hu) Inhibition 0.5 & 500 221
Spontaneous (Ha) Inhibition 0.025 171
SAMMA (Hu) No effect 10 5
FSP (Su) Inhibition 20 437
Nimodipine Cav3.2 (5.6 0.7)## rZP3 (Hu) Inhibition (50%) 3 282, 408##
Egg jelly (su) Inhibition (8) 287
Nisoldipine Cav1.2 (0.066)### High pH/depolarization Inhibition (6) 176, 540###
or ZP3 (Bu,Ram)
Cav3.2 (4.6 1.0)## FSP (Su) Inhibition 10 225, 408##
Nitrendipine Cav3.2 (8.8 2.3)## High pH/depolarization Inhibition (0.4) 176, 408##
or ZP3 (Bu,Ram)
PN200-110 Cav3 (0.04)* ZP (Mo) Inhibition (0.07) 11, 579*
High pH/depolarization Inhibition (0.1) 175
or ZP3 (Bu,Ram)
Verapamil Cav1 (50)* native ZP (Hu) No effect 20 52, 579*
Cav(0.4)*** rZP3 (Hu) No effect 10 & 80 5,*** 282
Cav3.2 (32.7 2.4)## Folicular Fluid (Hu) No effect 10 75, 408##
SAMMA (Hu) Inhibition (0.4) 5
High pH/depolarization Inhibition (0.8) 176
or ZP3 (Bu,Ram)
Spontaneous (Ha) Inhibition 0.01 171
FSP (Su) Inhibition 100 225, 298
Egg jelly (Su) Inhibition 81 456
-Agatoxin IVA Cav2.1 (0.020) rZP3 (Hu) No effect 0.06 282, 473
-Conotoxin GVIA Cav2.2 (1.5) rZP3 (Hu) No effect 0.002 90, 282
SNX-432 Cav2.3 (0.02) rZP3 (Hu) No effect 0.09 282, 385
Amiloride Cav3.2 (167) ZP (Mo) Inhibition 500 11
Diphenylhydantoin Cav3 (50)* SAMMA (Hu) Inhibition 210 5, 579*
Pimozide Cav3 (0.5)* native ZP (Hu) Inhibition 20 52, 579*
rZP3 (Hu) Inhibition 100 385
Progesterone (Hu) stimulate 10 & 20 209
ZP (Mo) Inhibition 1 11
Continued

Table 2.Continued
Calcium Channel Concentration Used, in M
Blocker Target (IC50, M) Inducer (Species) Effect in AR (IC50 for AR) Reference Nos.
Mibefradil Cav2.3 (0.4) ZP4 (Hu) Inhibition (1) 90, 120

Cav3.2 (0.25 0.06)## ZP3 (Hu) Inhibition (2.9) 120, 408##
native ZP (Hu) Inhibition 10 52
rZP3 (Hu) Inhibition (65%) 15 282
Progesterone (Hu) Inhibition 20 209
Ni2 Cav3.1 (312) ZP (Mo) Inhibition (50) 11, 313
Cav3.2 (13) rZP3 (Hu) Inhibition 14 282
Cav3.3 (195)
General calcium channel blocker
Cd2 Cav2.3 (0.8) ZP (Mo) Inhibition (375) 11, 90

Cav3 (245)
Co2 Cav1 and Cav2 (560)* High pH/depolarization Inhibition (110) 176, 579*
or ZP3 (Bu,Ram)
Cav3 (160-3,000)* FSP (Su) Inhibition 750 437
La3 Cav (15)* Egg jelly (Su) Inhibition 160 147, 579*
Ni2 Cav2.3 (27) High pH/depolarization Inhibition (60) 90, 177
or ZP3 (Bu,Ram)
Cav3.1 (312) SAMMA (hu) Inhibition (75) 5, 313
Cav3.2 (13) FSP (Su) Inhibition 200 437
Cav3.3 (195)
Intracellular calcium channel blocker
2-APB IP3-sensitive (42) SAMMA (Hu) No effect 50 5, 348

Thapsigargin 1 M Inhibition (40%) 50 5
(Hu)
Ruthenium red RyR (3) Egg jelly Inhibition 50 325
SOC blocker
AN1043 2
SOC (TG Ca rise: ZP3 (Mo) Inhibition 10 (1.1) 387, 391
0.7 0.2)
SKF96365 SOC (10) rZP3 (Hu) Inhibition 20 279
FSP (Su) Inhibition 2 256
Common calcium channels blockers used to inhibit the AR in different species (Bu, bull; Ha, hamster; Hu,
human; Mo, mouse; Ra, Ram; Su, sea urchin). The table shows the specific target and the IC50 for each
inhibitor, the AR inductor used, the inhibitor concentration tested, and whether there was AR inhihibition (the
percentage or the IC50 of inhibition is indicated, when available). SAMMA: contraceptive microbicide that
induces acrosomal loss in noncapacitated human spermatozoa.
inhibitory action of 6 on CaV3 channels could explain A) mRNA DETECTION. Mouse spermatogenic cells ex-
the observation that despite the strong expression of press several transcripts coding for CaV channels. In the
CaV3.1 and CaV3.2 subunit mRNA in adult ventricular case of CaV1 and CaV2 channels, RT-PCR experiments re-
myocytes, no CaV3 currents are detectable in these cells. vealed the presence of CaV1.2, CaV2.1, and CaV2.3 (164,
Postinfarcted ventricular myocytes often display reoccur- 324), while CaV2.2, CaV2.3, and CaV1.2 (219, 400) tran-
rence of CaV3 currents with higher expression of mRNA scripts were reported in mature human spermatozoa. Ad-
for CaV3.1 and CaV3.2 channels (115). ditionally, specific spliced transcripts for CaV1.2 are ex-
pressed in human sperm (219, 220). All three transcripts
1. CaV channels in spermatogenic cells and for CaV3 channels are present in mouse and human sper-
spermatozoa matogenic cells (164, 276, 400, 463, 474). With respect
to auxiliary subunits, transcripts for all four known
To establish the presence and activity of CaV channels in sper- genes encoding the subunits were reported in mouse
matozoa, researchers have used diverse experimental ap- spermatogenic cells (463) and 2, 1, 2, and 4 in
proaches to detect mRNA, protein, and currents (TABLE 1). mature human spermatozoa (282).

DARSZON ET AL.
B) PROTEIN DETECTION. Immunocytochemistry and/or West- recorded in either cell type, under the experimental condi-
ern blot experiments using specific antibodies for the differ- tions tested (137, 160). On the other hand, currents from
ent CaV subunits confirmed the presence of CaV1.2 and CatSper channels (see sect. IVD) have been consistently
CaV2.1 proteins in spermatogenic cells (463), and all the detected in mouse epididymal spermatozoa and not in the
members of CaV1 and CaV2 subfamilies, with the exception cells from CatSper null mice. These findings challenge pre-
of CaV1.1, CaV1.3, and CaV1.4, in mature sperm (514, 554, vious functional, molecular, and pharmacological data in-
556). All three CaV3 proteins were found both in mouse dicating the involvement of CaVs and other channels in
and human mature sperm (164, 276, 400, 463, 475, 514, diverse sperm functions (see discussion in sect. V, C and D)
556). Importantly, Western blot and immunohistochemis- and imply that the CatSper channel is one of the major
try experiments using CaV3.1 or CaV3.2 null mice as con- Ca2-conducting channels in spermatozoa (433).
trols corroborated that CaV3.2 channels are present in ma-
ture spermatozoa (160). Despite the presence of several CaV
proteins, the function of each of these channels has not been
B. Cyclic Nucleotide-Gated Channels
fully corroborated (see below).
Cyclic nucleotide-gated (CNG) channels have a well-estab-
C) CURRENT DETECTION. Patch-clamp studies, the classical ap-
lished role in sensory transduction processes (vision and

proach to examine cell currents, have not revealed CaV1 or olfaction); their function in other tissues such as heart, kid-
CaV2 (HVA) currents in spermatogenic cells, including ney, brain, or spermatozoa is not so clear (285, 353). CNG
those from CaV3.1 and 3.2 null mice (134, 160). However, channels function as heterotetramers resulting from the
channel antagonists to CaV1 or CaV2 channels interfere combination of A (CNGA1 4) and B (CNGB1 and 3) sub-
with certain sperm Ca2 responses (554) and the AR (46, units with a stoichiometry that varies in different tissues.
221), suggesting that these channels may be present in an Heterologous expression of CNG channels has shown that
homomeric A (with the exception of A4; Ref. 109) but not
electrophysiologically inactive state and activated during
B subunits can form functional channels (107). Each sub-
sperm differentiation and maturation.
unit contains six TM segments and a single cyclic nucleo-
tide-binding site near the COOH terminus (74, 285). CNG
In contrast, CaV3 currents have been well documented in
channels open upon cAMP or cGMP binding, have low ion
spermatogenic cells by patch-clamp experiments (14, 234,
selectivity, are weakly voltage sensitive, and unlike other
324, 449). These currents display the properties of somatic
channels, they do not inactivate or desensitize (74). De-
cell CaV3 currents, such as low voltage thresholds for acti-
pending on the particular subunit combination, they exhibit
vation and inactivation, and near-equivalent Ba2 selectiv-
differential selectivity to cAMP and cGMP (412). As CNG
ity relative to Ca2. The pharmacology of the currents is
channels are nonselective under physiological conditions,
also consistent with CaV3 channels as they are inhibited by
they usually carry inward Na and Ca2 currents (353).
the anticipated concentrations of amiloride, pimozide,
The first sperm ion channel cloned from mouse testis was a
mibefradil, kurtoxin, and low Ni2 concentrations (14, CNG channel (557); thereafter, others have been cloned
396) (TABLE 2). Drugs normally considered as CaV1 and (285). The A3 and B1 subunits were localized within the
CaV2 antagonists such as nifedipine and related 1,4-dihy- flagellum of mature mouse spermatozoa (285). Addition of
dropyridines (DHPs), at lower (M) concentrations can permeable cGMP to mouse spermatozoa provoked [Ca2]i
also block spermatogenic T-type currents (CaV3) (14, 449) increases dependent on [Ca2]e, while cAMP was less effec-
(TABLE 2). The pharmacological profile of the spermato- tive (295, 558). Curiously, spermatozoa from CatSper null
genic cell currents (137), as well as the current recordings mice do not present this response (432), and the A3 null
from CaV3.1 and Cav3.2 null spermatogenic cells (160), are mice are fertile (285). These observations throw into ques-
consistent with CaV3.2 being the principal isoform function the relevance of these channels in sperm physiology. In
tionally present in these cells. Additionally, CaV3 channels spite of this, their role in sea urchin sperm chemotaxis is
in spermatogenic cells appear to be regulated by protein well established (see sect. VI and Refs. 135, 284).
kinases (13), albumin (163), and CaM (336).
As mentioned before, patch-clamping mature sperm was C. Transient Receptor Potential Channels
extremely difficult (162) until Kirichok used the cytoplas-
mic droplet as the contact site for the pipette, which allows Initial evidence for the existence of transiet receptor poten-
giga-seal formation and electrical continuity with the cell tial (TRP) channels came from a Drosophila melanogaster
(292) (reviewed in Refs. 329, 433). Since then, several ion mutant, reported by Cosens and Manning back in 1969
channels have been detected in testicular and epididymal (103). These authors reported a particular phenotype pres-
mouse spermatozoa (137, 346, 381, 431, 450, 566). In- ent in the trp mutant fly, which exhibited defects in electro-
triguingly, CaV3 currents such as those recorded in sper- retinogram recordings. Exposure to constant light pro-
matogenic cells were detected in testicular but not in epidid- duced a sustained response in the wild type, whereas the
ymal sperm (137, 566). CaV1 or CaV2 currents were not trp-deficient fly displayed a transient response, producing

the name transient receptor potential or TRP (368). After in Ref. 307). The function of this domain is not quite clear;
20 years, Montell and Rubin (370) reported the sequence of it may have a role in PIP2 binding (a modulator of TRP
the trp gene, which was later confirmed to encode a novel channels, see below) (439), or participate in channel assem-
class of ion channels with homology to voltage-gated chan- bly (205) as well as in tetramerization and gating (206).
nels (361, 411). Since then, this family has been extensively
studied, and it is now well established that they are involved PIP2 can modulate activation or inactivation of several ion
in diverse cellular functions. channels such as members of the TRPC, TRPM, and TRPV
subfamilies, and it probably modulates other TRPs too
Members of the TRP channel family are considered to be (494). PIP2 is present primarily in the cytoplasmic leaflet of
polymodal cellular sensors playing important roles in vi- the plasma membrane but not in trafficking vesicles or or-
sion, taste, hearing, touch, olfaction, thermal perception, ganelles, and therefore, its abscence would silence the chan-
and nociception (307). They are quite versatile and diverse nels as they are being synthesized or in transit (168). Alter-
in their features, selectivity, gating, and regulatory mecha- natively, the activity of the channels could be controlled
nisms and depending on the species, an organism can ex- indirectly after PLC transient activation and PIP2 depletion
press as many as 30 different genes. Based on sequence from the plasma membrane (494). Furthermore, since TRP
similarity they can be divided in seven groups: 1) TRPC channels are sensitive to several stimuli (pH, temperature,

(canonical), 2) TRPV (vanilloid), 3) TRPM (melastatin), phosphorylation, mechanical and osmotic changes, etc.),
4) TRPA (ankyrin), 5) TRPML (mucolipin), 6) TRPP PIP2 can serve as a means of desensitization or sensitization
(polycistin), and 7) TRPN (only one member found in ze- to such factors (494).
brafish, worms, and flies but not in mammals). TRP sub-
units contain six transmembrane (TM) segments, with both D. Store-Operated Channels
the COOH and NH2 termini facing the cytosol. Different
biochemical and optical methods suggest that active chan-
Frequently, [Ca2]i rises result from a combination of Ca2
nels assemble as homo- or heterotetramers (307). Hetero-
release from intracellular stores followed by Ca2 entry via
multimers have been described for all TRP subfamilies ex-
plasma membrane Ca2 channels, a concept termed capac-
cept TRPA and TRPN (483, 531). The modulation and
itative Ca2 entry or store-operated channel (SOC) activity
structural properties of TRP channels are quite diverse; for
(422). Several research groups tested this notion primarily
example, three members of the TRPM subfamily possess an
using compounds that empty intracellular stores (thapsi-
enzymatic activity linked to the COOH terminus; one chan-
gargin and cyclopiazonic acid, etc.) and Ca2 fluorescence
nel exhibits an ADP-ribose pyrophosphatase, and two more
imaging techniques (421, 494). The first Ca2 current reg-
contain an atypical protein kinase (369), while others can ulated by store depletion was measured by Hoth and Penner
act as mechanical (157) or humidity sensors (333). The in mast cells (264) and was named ICRAC (Ca2 release-
localization of TRP channels is also varied; some reside in activated Ca2 current). It was a very small Ca2-selective
the plasma membrane (PM) and some in intracellular or- current, not voltage activated and inward rectifying.
ganelles, or both (155). In spite of these divergences, there ICRAC was found and studied in several cell types (263),
are certain features shared by several members of the TRP but its molecular identity and sensing mechanism re-
superfamily, and in general, they are believed to integrate mained unknown until recently. TRP channels were for a
signals, as the response to one agonist is modified by an- long time the best candidates to produce SOC activity
other. For example, the TRPV1 threshold for heat sensitiv- (and probably ICRAC).
ity (43C) decreases if pHi drops to 6. Its sensitivity to
endocannabinoids, anandamide, piperine (black pepper), The recent discovery of two protein families, namely, stro-
and allicin (garlic) is modified by the presence of ethanol, mal interaction molecules (STIM) sensors and ORAI chan-
nicotine, or changes in phosphatidylinositol 4,5-bisphos- nels, introduced a new concept for the molecular composi-
phate (PIP2) levels (531). tion of SOC activity and ICRAC currents. This discovery also
helped to explain the contradictory results obtained with
Most TRPs are relatively nonselective Ca2 channels with a the heterologous expression of some TRP channels that did
PCa/PNa 10, with some exceptions such as TRPM4 and not have CRAC characteristics yet displayed SOC activity
TRPM5 that are monovalent-selective and do not permeate (reviewed in Refs. 151, 421). However, some TRPC sub-
Ca2. In contrast, TRPV5 and TRPV6 are highly Ca2 units are still recognized as part of the SOC machinery, a
selective with PCa/PNa 100 (92). The TRP domain located notion supported by a wealth of experimental evidence gen-
at the proximal COOH-terminal end (25 amino acids) con- erated before the discovery of STIM and ORAI proteins
tains the TRP box (EWKFAR) once considered as a signa- (reviewed in Ref. 54). SOC activity caused by TRPC chan-
ture of TRP channels. This domain is only present in all nels seems to be cell or condition specific (314). On the
TRPC members, whereas TRPM, TRPN, and TRPV mem- other hand, there is also evidence for the interaction of
bers display a smaller consensus sequence. Only W is pres- TRPCs with STIM and ORAI, leading to the proposal that
ent in all TRP members in this particular domain (reviewed SOC activity requires a multicomponent complex (see be-

DARSZON ET AL.
low). Alternatively, it has also been considered that ORAI SOC activity requires the formation of a macromolecular
proteins are not channels, but auxiliary subunits that confer complex named SOCIC (store-operated calcium influx
particular properties to the SOC machinery, such as Ca2 complex) that includes additional players such as the sarco/
selectivity (322). Despite the discovery of STIM and ORAI, endoplasmic reticulum Ca2-ATPase (SERCA) and EB1
the exact molecular composition of the SOC machinery is (end tracking protein). The assembly (or disassembly) of the
still a matter of debate and an interesting area of research. complex regulates SOC activity and does not occur ran-
domly in the membrane, but in specialized domains known
1. ORAI and STIM as lipid rafts (524). This is a new and fascinating field that is
just beginning to unfold, for example, after store depletion
The STIM and ORAI protein families with two and three STIM1 recruits adenylate cyclase, thereby altering cAMP
genes, respectively, are essential components of SOC activ- levels (316). At least two different functions for STIM1 at
ity. STIM1 and 2 are membrane proteins located primarily the plasma membrane have been described: 1) a cell-cell
in the ER where they act as Ca2 sensors. STIM1 is a mul- interaction mediator via its NH2 terminal that protrudes
tidomain protein with a single TM segment, which senses extracellularly and also 2) as a modulator of arachidonic
the Ca2 store content using a classical two EF-hand motif acid-regulated channels, which are different from SOCs and
located luminally at the NH2 terminus. It then transmits the independent of store depletion (357).

information to ORAI channels via domains in the COOH
terminus and thus regulates SOC channel opening (314). At
resting conditions, STIM1 exists as a dimer with Ca2 E. Intracellular Ca2 Channels
bound to the EF-hand (Kd of 0.2 0.6 mM) (481). Deple-
tion of the ER causes Ca2 dissociation from the EF-hand IP3Rs and RyRs are the major intracellular Ca2 channels
and a rapid spatial reorganization of STIM1, reported as located primarily in ER-type intracellular stores. In addi-
oligomerization and/or translocation to the PM or to tion, two-pore channels (TPCs) were recently described as
ER-PM junctions (151, 281). This movement allows intracellular Ca2 channels located in acidic organelles
STIM1 to communicate with ORAI channels located at the such as lysosomes (199, 585, 586). The activity of intracel-
PM. Despite its very similar architecture to STIM1, STIM2 lular Ca2 channels depends on the action of second mes-
apparently does not translocate or oligomerize, and its role sengers produced by different stimuli and diverse signaling
during SOC activity is still controversial (281). ORAI chan- pathways (IP3, cADPR, and NAADP, respectively). The di-
nels were named after the three heavens gate keepers Eu- versity of these channels and their mobilizing agents allows
nomia (Order or Harmony), Dike (Justice) and Eirene the cell to produce specific signals due in part to spatiotem-
(Peace) in Greek mythology (172). They are small proteins poral differences in the signaling cascades.
(28 33 kDa) with four TM segments that tetramerize to
form the ion channel pore. ORAI proteins are highly glyco- 1. IP3Rs
sylated with cytoplasmic NH2 and COOH termini (436).
Coexpression of STIM1 with ORAI1, -2, and -3 reconsti- There are three genes coding for IP3Rs with differential
tutes CRAC currents with different properties in terms of tissue expression and distinct properties. The generation of
Ca2 and Na selectivity, current amplitude, and pharma- IP3 together with DAG occurs via stimulation of G protein-
cological sensitivity to drugs such as 2-APB (184). ORAI3 is coupled receptors (GPCR) or tyrosine kinase-coupled re-
activated by 2-APB independently of STIM1 or store deple- ceptors which activate PLCs that in turn cleave PIP2 to
tion. Low concentration of 2-APB (510 M) stimulates produce IP3 and DAG. The IP3R is regulated by phosphor-
STIM1-ORAI1 currents, but higher concentrations (50 ylation via Ca2/CaMKII and cGMP-dependent protein ki-
M) completely inhibit ORAI1 and partially inhibit ORAI2 nase (359). The three-dimensional structure of IP3R is te-
currents (184). Based on these properties, Goto et al. (222) trameric with three functional domains: the ligand-binding
developed 2-APB derivatives to differentially activate and domain (NH2 terminal), the channel-forming domain with
inhibit SOCs. The pharmacology of SOC activity began six membrane-spanning regions (COOH terminal), and the
before the discovery of ORAI channels; therefore, data in- modulatory/coupling domain, separating the two regions
terpretation and usage of these compounds can be compli- (6). There are several proteins that bind to IP3R and mod-
cated; there are excellent reviews concerning this issue (436, ulate its activity such as cytochrome c, huntingtin, CaM,
500). Homer, CARP (carbonic anhydrase-related protein), PKA,
and RACK1 (substrate for the Src protein-tyrosine kinase),
As mentioned before, the discovery of ORAI channels and among others (121, 359). This diverse set of proteins allows
the establishment of the identity of the ICRAC current did the production of unique spatiotemporal patterns in Ca2
not eliminate TRP channels as participants in SOC activity. intracellular signals (121). Particularly interesting is the
In fact, there is evidence that TRPCs can interact with 530-amino acid long protein named IRBIT (inositol-1,4,5-
STIM1, and it has been suggested that native SOC channels trisphosphate receptors binding protein released with IP3)
may be formed by a combination of STIM, ORAI, and that was recently described as an IP3R binding protein (6).
TRPC proteins (314). Moreover, Vaca (524) suggested that IRBIT acts as a pseudoligand (competitive inhibitor) be-

cause it does not activate IP3Rs and dissociates from them found in acidic organelles and was first described in plants
upon IP3 binding, regulating Ca2 signaling (6). After its (188) and in rats (274). Structurally, TPCs function pre-
release, IRBIT was found to interact and activate the pan- sumably as dimers; they resemble one-half of NaV and CaV
creatic but not the kidney Na/HCO3 cotransporter 1 channels, since they have two (instead of four) domains
(NBC1), suggesting a role in HCO3 secretion and pH reg- with the classical six TM regions. TPCs possibly represent
ulation in epithelial tissues (467). IRBIT has a multisite evolutionary intermediates of NaV and CaV channels (586).
strict phosphorylation requirement for its dual modulation Plant TPCs possess two EF-hand Ca2 binding motifs, a
activities (289). Regulation of HCO3 secretion and Cl feature lost in animal TPCs, consistent with their respective
absorption is an important but incompletely resolved issue regulation by Ca2 and nicotinic acid adenine dinucleotide
for different epithelia. For example, in the pancreatic duct, phosphate (NAADP) (404). NAADP was long known as a
it involves the concerted function of cystic fibrosis trans- potent Ca2-mobilizing agent (nanomolar range), but the
membrane regulator (CFTR) and SLC26 transporters, al- target remained elusive until TPCs were discovered. TPCs
though the molecular mechanism is still under study. IRBIT have just arrived to the experimental arena; therefore, there
has emerged as a potential coordinator of this interaction, is still much to be done to discover their properties, topol-
linking fluid and electrolyte transport with cAMP and Ca2 ogy, and modes of regulation. For example, recently Cal-
signaling (572). Recently, it has been shown that IRBIT can craft et al. (78) proposed a mechanism by which the Ca2

also regulate Na/H exchanger type 3 (NHE3) activity in signal generated by NAADP targeting lysosomal TPC2
kidney proximal tubules (245). Interestingly, although could be further amplified by CICR via IP3R. The initial
IRBIT mRNA was found to be ubiquitously expressed, the NAADP-induced Ca2 signal may be small, but precisely
highest levels were detected in brain, kidney, and reproduc- localized to couple with neighboring IP3R or RyR systems,
tive tissues such as testis (6). Members of the SLC26 family to trigger larger or global Ca2 changes, if a particular
as well as CFTR channels have also been reported in sper- threshold is reached. This coupling of signals provides a
matozoa from different species (117, 249). versatile and spatially organized regulatory mechanism for
cellular functions (78).
2. RyRs
Interestingly, cADPR and NAADP, the agonists of RyR and
There are three genes encoding RyRs with several splice TPCs, respectively, are synthesized by the same enzyme,
variants and differential expression levels depending on the ADP-ribosyl cyclases, a family of multifunctional enzymes
tissue. RyR1 and RyR2 are present in skeletal and cardiac that catalyze several reactions including the synthesis of
muscle, respectively; all three isoforms are present in brain cADPR and NAADP, as well as their hydrolysis (443).
(469). RyRs are very large proteins that form homotetram- These reactions are differentially regulated by several fac-
ers with four subunits of 565 kDa each, and the huge cyto- tors that include pH, cGMP, cAMP, Zn2, etc. (443).
solic NH2 terminal (80% of the protein) is a scaffold for
proteins that regulate channel function (303). The modula- It is worth mentioning that sea urchin sperm expresses a
tion of RyRs is quite diverse, including CaM, phosphoryla- particular type of NAADP synthase, and a possible role for
tion (PKA and CaMKII), oxidation, nitrosylation, ions NAADP has been proposed for the sea urchin AR (529,
(Ca2 and Mg2), pH, and several regulatory proteins (re- 530).
viewed in Ref. 580). In muscle cells, activation by Ca2
plays an important role, a mechanism known as Ca2-in- F. SOC (or SOCIC) Machinery Components
duced Ca2 release (CICR). In nonmuscle cells, RyR2 and in Spermatogenic Cells and Spermatozoa
-3 are also activated by Ca2 and/or by cADPR (580). The
diverse regulatory mechanisms allow the participation of 1. mRNA detection
RyRs in several cellular functions such as muscle contrac-
tion, exocytosis, secretion, neurotransmitter release, and As mentioned earlier, SOC channels have important roles in
gene transcription. During muscle contraction, there is a sperm motility and the AR (see sects. IVB and V). Accord-
well-known excitation-contraction coupling and physical ingly, the presence of different components of the SOC
interaction between CaV1 channels and RyR1. Similar in- complex has been reported in spermatogenic and sperm
teractions have been described in smooth muscle and neu- cells (TABLE 3). Mouse spermatogenic cells express tran-
rons that are probably mediated by the Homer protein. scripts for all three IP3Rs and RyRs (515). Additionally,
Interestingly, RyRs have been reported to interact with transcripts for trpc17 and trpc1, -3, -6, and -7 were found
other Ca2 channels such as members of the TRPC family in mouse and human spermatogenic cells, respectively (85,
that can also bind Homer (416). 516). More recently, several transcripts from other TRP
subfamilies [TRPV (V1, V5) and TRPM (M3, M4, M7)]
3. TPCs have been documented in rat spermatogenic cells (53, 319).
In particular, TRPV1 (boar) (49, 50) and TRPM8 (human
TPCs are encoded by either two or three genes, depending and mouse) transcripts have been found in spermatozoa
on the species (62). This novel class of Ca2 channels is (143, 347).

DARSZON ET AL.
Table 3. Expression of TRP and intracellular channels in sperm cells

Channel mRNA WB ICC Reference Nos.
IP3R Mo Mo Rt Ha Do Mo (h) Rt (h) Ha (h) Do (h) 515, 539

RyR Mo Mo (h,f) Hu (h) 119, 239, 515
TRPC1 Mo Hu Mo (f) Hu (f) 85, 516
TRPC2 Mo Mo (h) 283, 516
TRPC3 Mo Hu Mo (h,f) Hu (h,f) 85, 516
TRPC4 Mo 516
TRPC5 Mo 514
TRPC6 Mo Hu Mo (h,f) Hu (h,f) 85, 516
TRPC7 Mo Hu 85, 516
TRPM3 Rt 318
TRPM4 Rt 318
TRPM7 Rt 318

TRPM8 Hu Hu Hu (h,f) 143, 347
TRPV1 Rt Bo 49, 50, 319
TRPV5 Rt 318
The detection of the mRNA, the protein by Western blot (WB), or the subcellular localization (h, head; f,
flagella) by immunocytoche mistry (ICC) of each channel and different species (Mo, mouse; Hu, human; Rt, rat;
Bo, boar; Ha, hamster; Do, dog) is indicated. Detailed information about transient receptor potential (TRP)
knockout phenotypes can be found in Reference 562.
2. Protein detection the testis, although morphologically differentiated, mam-

malian spermatozoa are unable to swim or fertilize eggs.
IP3Rs have been immunolocalized to the acrosome of sev- Their suppressed ability to move forward, possibly related
eral species (539) and in some cases also detected in the to the changing levels of 2-arachidonoylglycerol through-
RNE (260). RyR3 protein was also found in mouse sper- out the epididymis, is recovered as they further mature (94)
matozoa (119, 239, 515). Several TRPC proteins were iden- (FIG. 2). In contrast, spermatozoa of invertebrates and
tified in mature mouse and human spermatozoa. TRPC1, lower vertebrates have full fertilizing capacity when re-
-2, -3, and -6 were immunolocalized predominantly in the leased from the testis (571). Transit from the epididymal
flagella, and TRPC2 in the overlying region of the mouse caput to the cauda via the corpus advances sperm functional
sperm acrosome (283, 516). In human spermatozoa, maturation and takes from 7 to 11 days depending on the
TRPC1, -3, -4, and -6 were detected in the flagella (85, 516). species. Different epididymal regions secrete factors that
From the newly discovered SOC components, only STIM1 mediate sperm maturation (100). For example, certain
has been immunolocalized to the neck region of human
mammalian cysteine-rich secretory proteins (CRISPS) are
sperm (105), although it is likely that ORAI proteins are
incorporated into, and onto, sperm during spermatogen-
also present and functional in spermatozoa.
esis and posttesticular sperm maturation. CRISPS con-
tain two domains, a CAP domain and a characteristic
There are also reports of the presence of TRP channels from
cysteine-rich COOH-terminal domain, referred to as the
other subfamilies. For example, western blot analysis veri-
Crisp domain (96, 214). Epididymal principal cells se-
fied the expression of TRPM4, TRPM7, and TRPV5 in rat
spermatogenic cells and spermatozoa (318), and TRPM8 crete high concentrations of CRISP1 and CRISP4 into the
expression was confirmed by western blot and immunocy- epididymal lumen where they surround the spermato-
tochemistry in human and mouse spermatozoa (143, 347). zoon. CRISPs have been found to regulate voltage-gated
The physiological role of these so-called sensory channels K channels (KV), CaV channels, CNGA channels, and
may be in sperm guidance processes such as chemo- or RyRs (96, 214, 570).
thermotaxis.
Epididymal fluids from several species seem to be hyperos-
molal (280 420 mmol/kgH2O) to blood serum that is
IV. MAMMALIAN SPERM MATURATION isotonic to female tract fluids (300 mmol/kgH2O) (574).
When ejaculated, spermatozoa mix with accessory gland
A. The Epididymis fluids that constitute most of the ejaculate and that are
hyposmolal to epididymal fluid. Therefore, ejaculated
General transcription and translation of sperm proteins sperm face a hyposmotic challenge upon ejaculation. In-
cease in the late spermatid stage in the testis. Upon leaving deed, it is worth emphasizing that spermatozoa develop

their ability for osmotic regulation while traveling through is the core structure that propels flagella (328). It is typically
the epididymis (101, 496). formed by nine microtubule doublets around a central pair
of microtubules (9 2 structure). Each outer doublet is
During posttesticular maturation, rodent spermatozoa take connected to its neighbor by nexin protein links (not
up small water-soluble components of the epididymal fluid shown) and also has a series of projections, called the radial
(i.e., taurine, L-carnitine, myo-inositol, and glutamate) spokes, that act as spacers to position the doublets in a circle
(522). Human spermatozoa seem to use inorganic os- around the central pair of microtubules. All doublets are
molytes instead (574). This process of slow isovolumetric anchored to the base of the flagellum, which usually termi-
regulation is likely to help spermatozoa contend with the nates in a centriole-like basal body or in the connecting
hyposmolal fluids of the male accessory glands and female piece in mammalian spermatozoa. The force that slides mi-
tract that they encounter upon ejaculation. crotubules producing flagellar beating is generated by the
axonemal motor protein dynein ATPases that walk unidi-
It is interesting that male infertility of apparently healthy rectionally towards the base of the flagellum. Dyneins from
animals able to mate has, in some instances, been correlated one doublet transiently interact with the following doublet.
with spermatozoa displaying angulated flagella when re- During the beat cycle, dyneins on one side of the axoneme
leased from the epididymis. Furthermore, several transgenic tend to bend the flagellum in one direction, and dyneins on

mice with normal sperm production that are infertile also the opposite side bend it in the other direction (reviewed in
display flagellar angulation after epididymal transit, which Ref. 72). Hence, the beat consists of alternate episodes of
is uncommon in normal mice (101). Angulation seems to activation of the two opposing dynein bridge sets, where
result from permeability problems and often occurs at the each set is switched on and the other off alternatively
end of the midpiece where the cytoplasmic droplet is located at appropriate mechanical set points. The control of the
(see sect. I for definition and FIGS. 1 and 2). The capacity of activation switch of the two dynein-bridge sets resides in the
mouse and human spermatozoa to properly regulate their axoneme itself (see Ref. 328 for a comprehensive review).
volume has been correlated with their ability to fertilize Dynein ATPase activity is known to be modulated by pH,
(173, 409). ATP, ADP, Ca2, and phosphorylation mediated for in-
stance by cAMP/PKA (123, 270, 271, 328, 372).
As the droplet is most likely a significant cytoplasm com-
partment, it is therefore an important site for osmolyte and 1. Adenylate cyclases in spermatozoa
water permeation. Morphological changes at this site min-
imize membrane damage due to stretching. In most species, cAMP is an essential second messenger in sperm physiology
the cytoplasmic droplet migrates from the interface be- (571) that is synthesized by two types of adenylyl cyclases
tween the head and flagellum to the annulus as spermatozoa (ACs): a soluble adenylyl cyclase (sAC) (330) and trans-
travel from the caput to the corpus of the epididymis (FIG. 2) membrane ACs (mACs, nine isoforms: mAC19). The sAC
(101). Irrespective of its position, the role of the cytoplasmic is regulated by bicarbonate and Ca2, and the mACs are
droplet in osmolyte regulation is consistent with the local- activated mainly by Gs (the -subunit of heterotrimeric G
ization of K and Cl channels involved in volume regula- proteins), and differently regulated by [Ca2], PKA, and
tion at this site in mouse and human spermatozoa (36, 573). PKC (236). The sAC is essential for sperm motility regula-
The droplet is usually not lost within the epididymis; it is tion and fertility (165, 252), is structurally and functionally
lost at ejaculation in many species though apparently not in similar to ACs from cyanobacteria, and is not activated by
humans (99). This loss seems to improve fertility in some G proteins or forskolin, the typical activators of mACs.
domestic species (101). Although in mouse sperm sAC is located in the flagellum
(252), it may affect plasma membrane changes in the ante-
rior part of the sperm head (189). In sea urchin spermato-
B. Motility zoa, it is distributed along the cell and also participates in
the AR (44). sAC was thought to function only in male germ
The fundamental goal of spermatozoa is to deliver their cells, but it is also present in somatic cells localized at dis-
genetic material to the female gamete. These highly special- tinct sites (169, 588, 589). In human airway cells, sAC is
ized cells have evolved sophisticated strategies to regulate localized to cilia where it regulates their motility (457).
their motility and succeed in finding the egg or oocyte. Thus
motility is one of the most important functions of male Initially it was thought that cAMP in spermatozoa was
gametes. Therefore, it is not surprising that sperm motility produced only by sAC and that Gs was absent. However,
defects have often been found to cause sterility in many evidence has suggested that several mACs and Gs may be
gene-targeted transgenic mice (PKA, CatSper, etc.) (165, present in sea urchin and mammalian spermatozoa (39, 44,
358, 390, 395, 459, 472, 542, 584), although they display 183, 478). Further experiments must corroborate that
no morphological defects. Most male gametes, including mACs are found in spermatozoa. There are experimental
some species of the plant kingdom, use a flagellum as the lines that indicate that both AC types are important for
driving force for motility. As seen in FIGURE 1, the axoneme sperm maturation, motility, and the AR. mAC3 null mice

DARSZON ET AL.
are subfertile as their spermatozoa show spontaneous AR the mechanism of how Ca2 controls the form of flagellar
alteration (334). bending is not fully understood. Pharmacological evidence
indicates that CaMKII and/or CaMKIV is involved in this
2. Flagellar form modulation by Ca2 process in mammalian sperm (269, 344). Recently, a novel
Ca2-binding protein, named calaxin, was identified as an
It is known that, in an appropriate medium supplied with axoneme component in sea squirt sperm (365). Bioinfor-
ATP, the sperm flagellum can be maintained motile after matics revealed that calaxin belongs to the neuronal cal-
removal of the plasma membrane by detergent treatment cium sensor protein family. Immunoelectron microscopy
(213). With the use of demembranated sea urchin sperm, and biochemical analysis showed that it interacts with the
the importance of Ca2 for sperm flagellar form was first dynein heavy chain at the outer arm dynein in a Ca2-
reported in 1974. The curvature of sperm swimming trajec- dependent manner. These results suggest that calaxin may
tories became larger as the Ca2 concentration was elevated be important for flagellar form regulation by Ca2 (365).
(73), due to the increase in flagellar asymmetry (71). These
discoveries led to a central dogma that the degree of flagel- C. Hyperactivation
lar beat asymmetry is determined by the global Ca2 con-
centration. The first Ca2 imaging of the moving sperm In mammals, spermatozoa become motile when ejaculated

flagellum was performed in 1993 using hamster spermato- into the female genital tract. The ejaculated spermatozoa
zoa (493). This experiment revealed that the [Ca2]i of show symmetric flagellar beating with low curvature and
hyperactivated spermatozoa was higher than that of acti- swim progressively with an almost straight path (FIG. 4).
vated spermatozoa (see sect. IVC). Hyperactivated sperm This swimming pattern is called activated motility. Fur-
motility occurs during capacitation (see sect. IVF) and is thermore, a subpopulation of sperm recovered from the
generally characterized by high amplitude and asymmetric oviduct shows vigorous asymmetric flagellar beating with
flagellar bends in nonviscous media (FIG. 4). However, we large amplitude and high curvature in a standard low-vis-
now know that the relationship between [Ca2]i and flagel- cosity medium, which is called hyperactivated motility.
lar asymmetry is not always proportional, as described later Hyperactivation is an essential process for spermatozoa to
in sea urchin and sea squirt (ascidians) spermatozoa (465, successfully fertilize oocytes, and Ca2 is a fundamental
562) (see sect. VI). Although several efforts have been made, regulatory factor; it is not known how it is triggered under
physiological conditions (reviewed in Ref. 488). Possibly
distinct factors such as bicarbonate (sect. IVD), progester-
one (sect. IVE), or a temperature decrease (sect. IVG) tran-
siently induce hyperactivation at different sites and occa-
sions while the spermatozoa search for the oocyte.
Ca2 is a fundamental regulatory factor for sperm hyper-

activation. With the use of demembraneted bull spermato-
zoa, it was demonstrated that Ca2 but not cAMP can
induce hyperactivation (259). For instance, 80% of sperma-
tozoa show hyperactivation at 400 nM Ca2. As already
mentioned, [Ca2]i is higher in hyperactivated hamster
spermatozoa (100 300 nM) than in activated spermatozoa
(10 40 nM) (493). Ca2 channels at the plasma membrane
(CatSper) and from internal Ca2 stores (IP3R and RyR)
seem involved in achieving hyperactivation (see below). In
addition to Ca2 channels, sperm [Ca2]i homeostasis is
controlled by several types of Ca2 transporters (552). A
plasma membrane Ca2-ATPase 4 (PMCA4), localized in
the principal piece of the flagellum, is crucial for sperm
FIGURE 4. Properties and physiological roles of hyperactivation.
A: activated (left) and hyperactivated (right) flagellar shape and mo-
function since its elimination affects sperm motility (459)
tility direction (arrows) displayed by spermatozoa in nonviscous ex- and hyperactivation (395) resulting in male infertility. Mi-
perimental media. Activated spermatozoa advance showing a sym- tochondrial abnormalities found in PMCA4-deficient sper-
metric flagellar bend, while hyperactivated spermatozoa tumble in matozoa (395) suggest Ca2 overload due to defective Ca2
one place and do not advance efficiently due to an asymmetric extrusion.
flagellar beating pattern. B: importance of hyperactivated sperm
motility: i) To advance in highly viscoelastic fluids in the female genital
tract more effectively than activated sperm, ii) to detach spermato-
1. Physiological roles of hyperactivation
zoa from the isthmus reservoir and advance towards the ampulla
(site of fertilization), and iii) to facilitate sperm penetration through Hyperactivated spermatozoa are less progressive than acti-
the cumulus matrix and the zona pellucida. vated spermatozoa in low viscous media. Some hyperacti-

vated cells are completely nonprogressive; they just tum- In contrast to CatSper1 4, auxiliary subunits were identi-
ble in the same place. In contrast, hyperactivated sper- fied by proteomics as CatSper1-associated proteins. Ex-
matozoa swim more progressively than activated pressing a fully functional HA-EGFP (green fluorescent pro-
spermatozoa in high viscous and/or high viscoelastic me- tein)-tagged CatSper1 in a CatSper1-null background, al-
dia (489, 491) (FIG. 4). This experimental observation lowed immunopurification of protein complexes containing
indicates that one of the physiological roles of hyperac- CatSper1 and novel proteins CatSper and CatSper (332,
tivation is to ensure progressive movement in the high 543). CatSper is a 126-kDa protein containing two TM
viscous fluids encountered in the female reproductive segments with two short cytoplasmic domains and a large
tract, such as the cervical mucus which is several hun- extracellular domain. CatSper is a 131-kDa protein con-
dredfold more viscous than aqueous experimental media taining a single TM segment with a large extracellular do-
(560). Hyperactivation is also important to facilitate sperm main and a short cytoplasmic tail. Notably, CatSper pro-
penetration of the extracellular matrix of cumulus cells and teins including CatSper and CatSper are practically ab-
of the oocyte, known as the zona pellucida (ZP) (FIGS. 3 and sent in mature spermatozoa from CatSper1-null mice (332,
4) (482). Certain transgenic mice are infertile due to sperm 543). This is strong experimental evidence that CatSper
motility defects, but in some cases their spermatozoa can and CatSper are auxiliary proteins for the CatSper chan-
fertilize ZP-free oocytes, but not intact oocytes (334, 426, nel. The large extracellular domains of CatSper and

432). This defect has been attributed to a specific inability CatSper suggest possible channel modulation by external
to initiate hyperactivated motility. On the other hand, it is signals such as ligands and cell-cell or extracellular matrix-
known that the head of mammalian spermatozoa attaches cell interactions during sperm maturation in the epididymis
to epithelial cells in the isthmus of the oviduct, a site known and/or the female genital tract. As indicated earlier, all
as the sperm reservoir (150, 492) (FIG. 3). Spermatozoa CatSper-related genes (except for CatSper) were found in
detach from the reservoir by hyperactivated motility and the sea squirt (Ciona intestinalis) and sea urchin (Strongy-
swim forward to the site of fertilization, the ampulla (150). locentrotus purpuratus) genomes, but not in those of flies
Therefore, a third function of hyperactivation is to allow (Drosophila melanogaster) or worms (Caenorhabditis el-
spermatozoa to detach from the sperm reservoir in the ovi- egans) (77, 332, 543). Just recently, CatSper, the newest
duct which may occur in multiple occasions (FIG. 4). auxiliary protein, was identified in the CatSper molecular
complex (124). CatSper has a single TM segment with a
large extracellular domain and a short cytoplasmic tail, a
D. CatSper Channels similar protein topology as CatSper. Notably, CatSper-
null transgenic male mice are infertile due to a hyperactiva-
1. Structure of CatSper tion defect. The amount of CatSper1 in spermatozoa is
remarkably reduced in the CatSper-null mice, indicating
that CatSper is an essential auxiliary subunit for expres-
A combination of bioinformatics and gene-targeted trans- sion of the functional CatSper channel (124).
genic mouse strategies revealed that spermatozoa possess a
sperm-specific cation channel named CatSper (cation chan- 2. Biophysical properties of CatSper
nel of spermatozoa), which is essential for hyperactivated
sperm motility. CatSper is a novel type of Ca2 channel In spite of intensive efforts by many researchers, functional
most likely composed of four separate pore-forming sub- CatSper channels have not been successfully expressed in
units, CatSper1 4 (9, 279, 335, 425, 432), and three addi- heterologous systems, preventing their full biophysical
tional auxiliary subunits, CatSper, CatSper, and characterization. They have only been studied in spermato-
CatSper (124, 332, 543) (FIG. 5). zoa of wild-type (WT) and mutant mice. Male infertility
was observed in CatSper1 4 gene-targeted transgenic mice
The pore-forming subunits of CatSper (CatSper1 4) struc- with an identical phenotype (278, 423, 426, 432), which
turally resemble CaV channels, including the selectivity con- indicates that all pore-forming CatSper genes are essential
sensus sequence for Ca2 (T/S X D/E X W) (335) (FIG. 5). for the functional channel. It seems likely that the CatSper
Each of the 4 separate CatSper subunits has 6 TM segments, heterotetramer must be composed of the four distinct sub-
similar to KV channels, in contrast to CaV channels which units, differing from other six TM channels such as KV,
are composed of 24 TM segments in a single polypeptide. In TRP, and CNG channels that are frequently composed of
all known voltage-gated cation channels, the 4th TM seg- homotetramers, although in some instances these latter
ments (S4) have several positively charged residues (R/K). classes of channels may also form heterotetramers under
This is the case of CatSper1 and CatSper2, although physiological conditions (285).
CatSper3 and CatSper4 have only two (335), which may
explain the mild voltage dependence of its gating. Remark- The recording of CatSper ionic currents necessitated the
ably, CatSper1 has a histidine-rich large cytoplasmic termi- introduction of a strategy to facilitate obtaining whole cell
nal domain (432) that varies in sequence and length among patch-clamp recordings. Kirichok et al. (292) found that the
species, even among primates (414). cytoplasmic droplet (see FIG. 1) of epididymal sperm is a

DARSZON ET AL.

FIGURE 5. Structure of CatSper channel. A, left: protein topology of CatSper1 channel showing six trans-
membrane segments (S1-S6) with S4 containing several positively charged residues, the pore loop, and
cytoplasmic COOH and NH2 termini; the latter has a histidine (His)-rich region. Middle: Catsper 2, 3, and 4
have the same CatSper1 architecture except for a shorter COOH and NH2 termini, lacking the His-rich region.
Right: topology of the , , and CatSper subunits with one, two, and one trasmembrane segments,
respectively. B: sequence of the mouse CatSper1 NH2-terminal showing the histidine-rich region (51 of 250
histidine residues are shown in bold). C, top: amino acid sequence alignment of the human (h) and mouse (m)
S4 segment of CatSper 1 4, the putative voltage-sensor. Bottom: the S4 segments of each domain (I-IV) for
mouse CaV3.1 channel is also shown for comparison. Positively charged residues (arginine and lysine) are
shown in bold. D, top: amino acid sequence alignment of the human (h) and mouse (m) pore region of
CatSper1 4. Bottom: the loop forming sequence of each domain (I-IV) for mouse CaV3.1 channel is also shown
for comparison. The pore consensus sequence for Ca2-selective channels is [T/S]x[D/E]xW (shown in bold).
suitable region to form giga-seals with the patch pipette tions containing 2 mM Ca2 and with pipette solutions at
(292). In WT mouse spermatozoa, these authors recorded a pH 8. The Ca2 current was much smaller than the mon-
large current in divalent cation-free (DVF) medium, that ovalent cation current observed in the DVF medium and
was absent in spermatozoa from CatSper1-null mice. This was absent in spermatozoa from CatSper1-null mouse, in-
large monovalent cation current recorded in epididymal dicating its identity with ICatSper.
spermatozoa was defined as ICatSper. A striking feature of
ICatSper is that it is potentiated by intracellular alkalinization As for Ca2-selective channels, Ba2 can pass through
(292). Since the NH2-terminal cytoplasmic domain of CatSper channels without causing Ca2-dependent inacti-
CatSper1 is histidine rich, it is speculated that this domain vation (292). With the use of Ba2 tail currents, it was
functions as a pH sensor. observed that ICatSper is much less voltage-dependent than
typical voltage-gated channels. It is worth pointing out that
The size of the monovalent ICatSper was reduced by increas- the voltage at which ICatSper can be activated shifts toward
ing extracellular Ca2 concentrations with an IC50 of 65 negative potentials, closer to sperm Em values, as the pipette
nM (292), a typical property of Ca2-selective channels. pH is raised from 6.0 to 7.5 (292), the physiological range
Ca2 inward currents were recorded with external solu- for sperm pHi. This property reasonably explains why

ICatSper is stimulated by intracellular alkalinization. It was combine to establish the importance of hyperactivation
previously reported that a medium of high K and pH 8.6 for fertilization and the key involvement of CatSper in
(K8.6) induced an increase in [Ca2]i in mouse spermato- these motility-related processes.
zoa (554). This cell-depolarizing and -alkalinizing stimulus
is an accidentally appropriate condition to activate CatSper In addition to their role in Ca2 signaling, capacitated
channels. Indeed, this stimulus does not increase [Ca2]i in CatSper1-null mouse spermatozoa have been found to have
CatSper1-null mouse spermatozoa (83). higher cAMP levels (82) and lower ATP levels (565) than
WT sperm. It is not clear how these changes occur. Consid-
Electrophysiological recordings (292) as well as immunolo- ering that glycolysis is fundamental for mammalian sperm
calization of CatSper1 (432), CatSper (332), CatSper ATP synthesis (358) and that glycolytic enzymes are local-
(543), and CatSper (124) locate this channel in the princi- ized in the principal piece of the flagellum, CatSper might
pal piece of the flagellum. Furthermore, the NH4Cl-induced participate in regulating glycolysis. In demembranated bo-
Ca2 increase commences in the principal piece of the fla- vine spermatozoa, ATP dose-dependently potentiates high
gellum and spreads relatively slowly to the entire mouse WT flagellar curvature, indicating its importance in hyperacti-
spermatozoa, but not in CatSper1-null mouse spermatozoa vation (259). It is therefore clear that CatSper is essential for
(565). hyperactivation due to its influence on Ca2 influx and

indirectly on ATP levels. On the other hand, the lack of
3. Physiological functions of CatSper
CatSper does not reduce tyrosine phosphorylation during
capacitation (see sect. IVF) (83, 426) or interfere with the
The unquestionable importance of CatSper channels is as-
certained by the fact that null mice lacking any of the AR (see sect. V) (565).
CatSper isoforms are infertile (278, 423, 426, 432), and
human CatSper1 and -2 mutations have been associated 4. Regulation of CatSper under
with asthenoteratozoospermia (19, 20). physiological conditions
CatSper 1-null mouse spermatozoa cannot fertilize ZP- Cyclic nucleotides are known to induce an increase in
intact oocytes but do fertilize ZP-free oocytes, indicating [Ca2]i in WT spermatozoa (295), but not in CatSper1-null
that CatSper channels are required to allow sperm to spermatozoa (432). However, cyclic nucleotides do not di-
penetrate ZP (432). A careful comparison of the flagellar rectly activate CatSper channels (292). Considering that
beating form of WT and CatSper1-null spermatozoa re- ICatSper is potentiated by alkalinization, cAMP might in-
vealed that mutant spermatozoa were unable to display directly stimulate CatSper by a pHi increase (381, 433)
hyperactivated motility; their flagellar beating remains (FIG. 6). Uniquely, spermatozoa possess a Na /H ex-

symmetric with small amplitude and low curvature even changer (sNHE) (542) containing a consensus cyclic nucle-
in capacitating medium (see sect. IVF) (83). In agreement otide binding domain (CNBD) sequence. In addition, sNHE
with the involvement of CatSper1 in motility, CatSper2-
seems to directly interact with sAC (541), and both sNHE
null mouse spermatozoa swim less progressively than
and CatSper are localized in the principal piece of the fla-
WT mouse spermatozoa in a high viscous medium (426).
gellum (432, 542). With all information taken into account,
Observations 1 h postcoitum of sperm migration in the
it is likely that a functional link between sNHE and CatSper
oviduct documented the presence of both WT and
exists, a matter worth exploring. There is as yet no experi-
CatSper1-null spermatozoa in the lower isthmus, the
mental evidence that cAMP elevates sperm pHi, and it has
sperm reservoir in the oviduct (262). However,
CatSper1-null spermatozoa did not reach the ampullary- not been possible to functionally express sNHE in heterol-
isthmic junction, the upper region of the oviduct where ogous systems efficiently, impeding its direct characteriza-
fertilization takes place, even 4 h postcoitum (262). In tion. Future studies will surely determine how sNHE is
vivo video-recordings revealed that WT spermatozoa in regulated and its functional connection to the CatSper
the isthmus migrated toward the upper region of the channel.
oviduct by repeatedly detaching and reattaching to epi-
thelial cilia using vigorous flagellar beating. In contrast, It is worth noting that CatSper3,4-null spermatozoa keep
CatSper1-null spermatozoa remained attached to the ep- moving in Ca2-free medium (10 nM) (278), in which
ithelial cilia (262). These findings show that hyperactiva- mammalian spermatozoa are known to lose their motility.
tion is essential for sperm migration in the oviduct, par- This observation can be explained by the massive Na in-
ticularly to escape from the sperm reservoir. CatSper-null flux through CatSper channels that occurs in the absence of
mouse spermatozoa manifest clear defects in all physio- external Ca2 in WT spermatozoa (511), but not in
logical roles previously proposed for hyperactivation CatSper-null spermatozoa (80). In mouse (161) and human
(FIG. 4): 1) progressive swimming in high viscous fluid, 2) spermatozoa (511), it was demonstrated that external Ca2
penetration through egg extracellular matrix, and 3) es- removal induces a Na-dependent depolarization and repo-
cape from the reservoir in the isthmus. These findings larization by readdition of Ca2.

DARSZON ET AL.

FIGURE 6. Ion fluxes during mammalian sperm hyperactivation and capacitation. Both processes take place
during sperm transit through the female tract and have parallel but also intercrossing pathways. Albumin
present in the female tract removes cholesterol from the sperm plasma membrane modifying several mem-
brane properties; it may also directly activate CatSper channels, increasing [Ca2]i. Activation of a Na/HCO3

(NBC) cotransporter (possibly activated by external Ca2) increases HCO3 levels activating a soluble adenylyl
cyclase (sAC) and producing cAMP. This cAMP may activate the sperm Na/H exchanger (sNHE) and
together with the activation of the proton channel (HV) (by Zn2 removal) would raise pHi, another cellular
parameter that activates CatSper and SLO3 channels. The overall Ca2 increase may influence glycolysis and
the axoneme activity promoting hyperactivation of motility. Several Ca2 mobilizing pumps (PMCA4, SPCA1)
and channels [IP3 receptor (IP3R), ryanodine receptor (RyR)] may also participate during Ca2 signaling.
Concomitantly, the increase in cAMP levels activates PKA, which after several unknown steps stimulates
tyrosine kinases to produce the tyrosine phosphorylation associated with capacitation. Possible connections
between hyperactivation and capacitation signaling cascades are indicated. In some species, capacitation is
accompanied by membrane hyperpolarization; the channels and transporters involved during this process are
indicated.
5. HVs may modulate pHi and CatSper somehow different in the two species. In mouse, sNHE
(see previous section) could dominate sperm pHi regula-
The regulation of pHi is fundamental for sperm function; tion rather than the HV. These differences may be related
in mammals, these cells face significant external pH to the fact that human spermatozoa do not seem to hy-
changes during maturation and in their transit through perpolarize during capacitation according to fluores-
the female genital tract (571). Recently, it was reported cence sperm population measurements (216), although
that voltage-gated proton channels (HVs) are function- flow cytometry results have indicated a small hyperpo-
ally expressed in mammalian spermatozoa (329). Inter- larization may occur (70). As indicated earlier, seminal
estingly, sperm HV currents are much larger in human fluid contains a high Zn2 concentration (mM), and mi-
than in mouse, suggesting that pHi regulation may be cromolar concentrations of this divalent cation block HV

channels (329). Zn2 concentrations are lower in the inhibitor, U73122, did not affect the progesterone-induced
female genital tract; therefore, the levels of this cation increase in [Ca2]i, indicating that PLC is not involved in
may contribute to the regulation of sperm responsiveness the signaling cascade (4). However, progesterone has been
at different stages by affecting HV activity. In addition, reported to stimulate PLC activity and hence IP3 production
lipids such as arachidonic acid potentiate HV activity (506). Reexamination of these matters is necessary. Re-
(329). It has been proposed that the depolarization- cently, a caged progesterone analog was developed (290).
induced [Ca2]i increase in human spermatozoa may be This compound is a promising tool to unveil the signaling
due to CaV channels (327). Alternatively, a depolarizing mechanism and physiological function of this steroid.
stimulus could increase pHi through the HV channel,
which would result in CatSper channel activation (433). The physiological function of progesterone had been as-
cribed mainly to the AR (see sect. V). Recent observations
E. Progesterone Induces Flagellar indicate that this steroid is also important for the modula-
Responses and Ca2 Release From tion of sperm motility. Progesterone induces [Ca2]i oscil-
Ca2 Stores lations in a fraction of human spermatozoa (10 36%), and
these responses are accompanied by increases in flagellar
bend amplitude (239), one of the important characteristics

Progesterone, a steroid hormone, is synthesized by the cu-
mulus/granulosa cells and regulates the female sexual cycle. of hyperactivation. While the initial Ca2 transient induced
This steroid was identified as the major active component of by progesterone is caused mainly by Ca2 influx, the sub-
the human follicular fluid that induces the AR (see sect. V) sequent Ca2 oscillations are likely due to Ca2 release
(398). While genomic action of this steroid is established from Ca2 store(s) localized in the base of the flagellum
through the nuclear receptor (167), progesterone binds to (239) (FIG. 6). Once the Ca2 oscillations started, removal
an unidentified receptor on the sperm plasma membrane, of external Ca2 (5 M) did not affect the oscillations,
provoking a rapid increase in the [Ca2]i (57), referred to as although further addition of a Ca2 chelator inhibited
a nongenomic action (337). The signaling mechanisms in- them. Low concentrations (50 100 M) of ryanodine ac-
volved in the nongenomic progesterone response in mam- celerated the oscillations, while high doses (500 M) re-
malian spermatozoa remain ill-defined. The progesterone- duced them. TMB-8 and tetracaine, inhibitors of Ca2 re-
induced [Ca2]i increase depends on extracellular Ca2 lease from Ca2 stores, arrested the oscillations, suggesting
(55), although Ca2 release from intracellular store(s) may the importance of store emptying/refilling cycles in this pro-
be involved (41). Stopped-flow fluorometry revealed that cess (238). The progesterone-induced oscillations are rela-
progesterone-induced [Ca2]i increases occur with a mini- tively insensitive to thapsigargin or cyclopiazonic acid, in-
mal time lag (50 ms) (290), indicating that the Ca2 trans- hibitors of SERCA, but sensitive to bis-phenol, a nonspe-
porter should be regulated by the receptor via a small num- cific Ca2-ATPase inhibitor. Western blotting detected
ber of intermediary steps, in contrast to the response to secretory pathway Ca2-ATPase (SPCA1) in human sper-
chemoattractants (200 ms time lag) in echinoderm sperma- matozoa, but not SERCA, and immunostaining showed
tozoa (286, 388) (see sect. VI). Nevertheless, the molecular that SPCA1 is localized in the head-tail junction, where the
identity of the Ca2 channel involved in the progesterone- Ca2 oscillations are observed (238). A fluorescently la-
induced rapid Ca2 influx is still unknown (see NOTE ADDED beled ryanodine binds to the same sites (238). These results
IN PROOF).
strongly suggest that SPCA1 is involved in refilling Ca2
during the Ca2 oscillations. Similar Ca2 oscillations were
A G protein-coupled receptor for progesterone was discov-
observed in resting conditions in a smaller fraction of the
ered in fish oocytes in 2003 (587), and expression of a
sperm population (293, 386), and this fraction increased
homologous membrane progesterone receptor (mPR) was
upon the addition of nitric monoxide (341) or two CaV1
detected in human testis (587). Western blotting revealed
mPR in human sperm membranes and immunostaining agonists, BAY K 8644 or FPL64176 (293). Therefore, the
localized this protein mainly in the midpiece of the sperm Ca2 oscillations might be induced by different types of
flagellum (507). Progesterone treatment of sperm mem- stimuli independently of their signaling mechanisms. The
branes increases GTPS binding, suggesting it activates a G progesterone-induced Ca2 oscillations occurring in a
protein (507). These findings point to mPR as a candidate sperm fraction of WT mouse spermatozoa are not observed
nongenomic receptor for progesterone in human spermato- in PLC4-null mice, suggesting the involvement of this PLC
zoa. Progesterone induced higher [Ca2]i transient in- in the Ca2 oscillations (186).
creases in capacitated than in noncapacitated human sper-
matozoa (see sect. IVF) (511). Phosphodiesterase inhibitors It is believed that mammalian spermatozoa have at least
potentiated this transient increase in noncapacitated sper- two Ca2 stores: the acrosome and the RNE (105). Calre-
matozoa and a PKA antagonist inhibited the response, sug- ticulin, a Ca2 binding protein generally found in Ca2
gesting that a PKA-dependent phosphorylation upregulates stores, and IP3Rs were detected in these two stores in sper-
the progesterone signaling cascade (512). In contrast, a PLC matozoa of various mammal species (260, 261, 377, 539).

DARSZON ET AL.
Interestingly, thapsigargin (a Ca2-ATPase antagonist) and 397), which in turn activate PKA, resulting in the rapid
thimerosal (IP3R and RyR agonist) can induce hyperactiva- phosphorylation of a subset of proteins (7, 242). As ex-
tion in bull spermatozoa (260). Ca2 imaging revealed that pected, the ion concentration changes experienced by sper-
these reagents immediately induce [Ca2]i increases at the matozoa as they enter the female tract alter their Em (i.e.,
base of the sperm flagellum, where the RNE is localized Ref. 148).
(261). The effects of thapsigargin and thimerosal are inde-
pendent of external Ca2 (261). Thimerosal can induce Two crucial ionic parameters for capacitation are [Ca2]i
hyperactivation even in CatSper2-null spermatozoa (345), and pHi (571), and both parameters increase during this
indicating the importance of Ca2 release from the Ca2 process (29, 141, 192, 329, 402, 583). It is known that
store to modulate the flagellar bend. On the other hand, among other components, Ca2, serum albumin, and
procaine, a reagent which triggers hyperactivation only in HCO3 are necessary for sperm capacitation in vitro
the presence of extracellular Ca2, failed to induce hyper- (538, 571). In mouse spermatozoa this process requires a
activation in CatSper2-null spermatozoa (345), indicating minimum [Ca2]e of 100 200 M (182, 343), although
that the procaine effect on sperm motility is mediated by 2 mM [Ca2]e is used in capacitating medium. Interesting
CatSper channels. results have shown that in addition to its critical role as
second messenger, Ca2 can act as a first messenger, and

a CaSR has been cloned (61). The presence of such a
F. Capacitation
receptor and its involvement in the HCO3-induced accel-
eration of flagellar beat frequency has been postulated in
After ejaculation into the female reproductive tract, sper- mouse spermatozoa (81).
matozoa must further mature, through a process referred to
as capacitation, before they can fertilize the egg (18, 112,
It is thought that albumin is needed to remove cholesterol
571). Although five decades have gone by since its discov-
from the sperm plasma membrane to induce its reorganiza-
ery, the molecular mechanisms of this intricate process are
tion and change its fluidity (108, 513, 538). These albumin-
still not well understood. Recent findings indicate that the
induced sperm plasma membrane changes could modulate
functional changes involved in capacitation result from a
Ca2 and/or HCO3 ion fluxes leading to the activation of
combination of sequential and in some instances parallel
sAC and increases in cAMP levels (538) (FIG. 6). Albumin
processes (7, 190). In light of this, capacitation can be
can act independently of the cAMP pathway by inducing
viewed as happening in both a faster and slower track,
proline-directed serine/threonine phosphorylation (277).
possibly involving distinct events in the sperm head and
Notably, this protein can also induce rapid (seconds)
flagella (534). Fast events such as sperm motility initiation
(81) and modification of membrane lipid architecture and [Ca2]i increases that were recently shown to depend on
organization (190) begin when spermatozoa are released CatSper and partially on pHi, and to be independent of G
from the epididymis and are exposed to increased HCO3 proteins and PLC (566). However, electrophysiological re-
concentrations. Slower events occur with a delay and are cordings revealed that albumin actually hyperpolarizes cor-
responsible for endowing spermatozoa with the ability to pus epididymal spermatozoa instead of depolarizing them
fertilize the egg, such as the acquisition of hyperactivated (566), an interesting finding worth further study. Here
motility (see sect. IVC) and the capacity to undergo the again we find that albumin, a component important for
physiologically relevant AR (see sect. V). capacitation, causes fast and slow responses in spermato-
zoa.
As spermatozoa travel from the epididymis and through the
female tract, they encounter significant changes in osmolar- A distinguishing feature associated with sperm capacitation
ity and in the concentrations of various ions. After mixing is the increase in protein tyrosine phosphorylation. Interest-
with the seminal fluid, stored spermatozoa in the caudal ingly, under capacitating conditions, mouse spermatozoa
epididymis are ejaculated into the female tract where extra- first reach maximum cAMP levels in 60 s and soon after
cellular [K] ([K]e) is reduced, and extracellular [HCO3] PKA-dependent phosphorylation occurs (446). Thereafter,
and [Na] are significantly increased. HCO3 concentra- at least 30 min must pass to detect tyrosine phosphorylation
tions elevate from 4 mM in the cauda epididymis (16) to (536). As these tyrosine phosphorylation events occur later
20 mM in the seminal plasma and female tract (340, 393). and are blocked by PKA inhibition, they are mediated by
These [HCO3]e increases immediately stimulate AC (393) cAMP and PKA (536). These initial findings in mouse sper-
and mouse sperm flagellar beating and enhance the cells matozoa (536) have been corroborated in a wide range of
sensitivity to depolarization by external K (553). Elevated species (193, 309, 535).
HCO3 also triggers a rapid decrease in sperm membrane
phospholipid asymmetry mediated by scramblases that ex- Recent reports have suggested that the Src family of pro-
ternalize phosphatidylethanolamine and phosphatidylser- tein tyrosine kinases (SFKs) mediate the capacitation-
ine (189, 241). The evidence indicates that HCO3 stimu- associated increase in tyrosine phosphorylation, hyper-
lates sAC (330) and increases cAMP levels (7, 68, 144, activation, and the AR in mammalian spermatozoa (166,

308, 364, 528). However, newly found evidence using pHi, and [Ca2]i changes necessary to achieve this matura-
Src-null mice and inhibitors of SFKs and phosphatases tional process. Bovine and mouse spermatozoa hyperpolar-
revealed that c-Src is either not important for these pro- ize during capacitation (12, 148, 374, 582). Noncapaci-
cesses or that the lack of c-Src can be compensated by tated mouse spermatozoa are relatively depolarized (Em ap-
other members of the SFKs. More importantly, these new proximately 35 to 45 mV) and hyperpolarize to
findings indicate that capacitation is regulated by two approximately 70 mV during capacitation (12, 148, 374).
parallel pathways. One of them requires activation of Voltage-gated K channels (447), Ca2-activated K chan-
PKA, and the other involves inactivation of Ser/Thr phos- nels (110), and inwardly rectifying K (Kir) channels (3,
phatases (299). 170, 374) have been detected in testis, spermatogenic cells,
and spermatozoa using molecular, electrophysiological,
As indicated above, capacitation in mammalian spermato- and biochemical tools. There is pharmacological evidence
zoa is HCO3 dependent. Aside from activating sAC (330), that Kir channels could contribute to the capacitation asso-
HCO3 influx hyperpolarizes mouse spermatozoa in a ciated with hyperpolarization (3, 374).
[Na]e-dependent manner (148). The ionic dependence of
this hyperpolarization is consistent with the presence of an Importantly, the second case of infertility caused by the
electrogenic Na/HCO3 cotransporter in these cells that is knockout of an ion channel, SLO3, was recently reported

important for capacitation. Furthermore, HCO3 uptake (450). The SLO3 channel belongs to the high-conductance
may contribute to the known pHi increase observed during slowpoke (Slo) K channel family and is only found in
sperm capacitation (148, 402, 583). mammalian spermatogenic cells and spermatozoa. It was
cloned a while ago from a mouse testis library and hetero-
Regulation of sperm pHi is fundamental for motility, capac- geneously expressed (458). The mouse SLO3 channel is
itation and the AR. Spermatozoa possess a specific Na/H activated by voltage change and intracellular alkalinization
exchanger, sNHE, that is a member of the mammalian and displays a K/Na permeability ratio (PK/PNa) of 5.
NHE superfamily (84, 427, 542). The sNHE protein is pre- Navarro et al. (380) reported a pHi-sensitive current in the
dicted to contain 14 TM segments and is located on the principal piece of epididymal mouse sperm, sharing some of
principal piece of the sperm flagellum. sNHE contains a the SLO3 characteristics, which they named KSPER. When
distinctive consensus sequence for a putative cyclic nucle- expressed in Xenopus oocytes, SLO3 is also stimulated by
otide-binding domain near its intracellular COOH termi- elevated cAMP levels through a PKA-dependent phosphor-
nus and four putative TM segments analogous to the volt- ylation. Patch-clamp recordings in testicular mouse sper-
age-sensing domain of voltage-gated ion channels (87). matozoa revealed that a K current that is time and voltage
These unique features suggest that sNHE may be regulated dependent is activated by intracellular alkalinization, has a
by cyclic nucleotides and Em (541), in addition to phos- PK/PNa 5, is weakly blocked by TEA, and is very sensitive
phorylation changes and protein interactions (84). sNHE to Ba2 (346). These currents are absent in testicular sper-
null male mice are infertile, and their spermatozoa are im- matozoa from Slo3 null mice, confirming their identity. Un-
motile (542). Addition of NH4Cl or permeable analogs of der capacitating conditions, spermatozoa from Slo3 null
cAMP (541) rescues these sperm functions. It turns out that mice are unable to perform progressive motility, to hyper-
full-length expression of sAC and its proper regulation by polarize, and to undergo the AR. Surprisingly, not even
HCO3 require sNHE. sNHE and sAC are apparently part Ca2 ionophore treatment can trigger the AR in these sper-
of a transduction complex that could efficiently regulate matozoa. These findings indicate that SLO3 channel activa-
pHi, HCO3, cAMP, and sperm motility (541). tion plays a preponderant role in the capacitation-associ-
ated processes necessary for fertilization; this channel is
As mentioned in section IVD, human spermatozoa pos- thus an attractive candidate for a male contraceptive drug
sess a high density of HV channels in the flagella while (450). Very recently, it was shown that SLO3 channels het-
those of mouse have less (329). This suggests that sNHE erologously expressed and in epididymal mouse spermato-
is more important for mouse sperm pHi regulation. This zoa are stimulated by PIP2. Epidermal growth factor (EGF)
exchanger may be stimulated by the hyperpolarization acting through the EGF receptor inhibits SLO3 currents in a
that occurs when these cells undergo capacitation. HV PIP2-dependent manner (501). There are some discrepan-
channels appear to be stimulated during capacitation, cies regarding the selectivity and voltage dependence be-
possibly by PKC-dependent phosphorylation (329), and tween SLO3 channels expressed in Xenopus oocytes and in
may contribute to the increase in pHi which is important spermatozoa that have to be worked out to fully understand
for this process. the contribution of SLO3 to the capacitation-associated hy-
perpolarization.
In addition to HCO3, Ca2, and albumin, capacitation in
vitro depends on the presence of K, Na and Cl in the Other ion channels and transporters may contribute to the
medium (7, 139). We still do not fully understand which capacitation-associated hyperpolarization. Amiloride-sen-
sperm ion channels and transporters are involved in the Em, sitive epithelial Na channels (ENaCs) have been reported

DARSZON ET AL.
to be present in mouse spermatozoa. Noncapacitated Many important questions remain unanswered about the
mouse spermatozoa are depolarized by the addition of ex- complex process of capacitation; some related to Ca2 are
ternal Na and hyperpolarized by ENaC blockers and by a 1) as spermatozoa from CatSper null mice undergo the nor-
cAMP/PKA-dependent process (248, 297). ENaCs and mal changes in tyrosine phosphorylation and the AR, this
CFTR have been found to colocalize in several cell types channel seems unnecessary for capacitation, but needed for
where they regulate each other (48). CFTR is a cAMP- hyperactivation. If Ca2 influx is needed for capacitation,
modulated, ATP-dependent, Cl channel, and many of its other Ca2 transporters must participate in this process and
mutations cause cystic fibrosis (CF), the most prevalent hu- in the AR. What is their identity and modes of regulation?
man genetic disease (438). ENaCs and CFTRs have been 2) Are any of the proteins important in Ca2 transport
reported to be present in the midpiece of mouse and human during capacitation regulated by their phosphorylation
sperm flagella (249, 569). Experimental evidence suggests state? 3) What is the interplay between Em, pHi, [Ca2]i,
and cAMP? 4) Do the lipid changes associated with capac-
that cAMP induced activation of CFTR leads to the closing
itation regulate Ca2 transport?
of ENaCs, contributing to the capacitation-associated hy-
perpolarization (249).
G. Other Aspects of Mammalian
Cl can be transported across the plasma membrane by

Sperm Motility
different Cl channels, exchangers, and cotransporters; it is
important for the regulation of cell Em, volume, pHi, and 1. Chemotaxis
HCO3 transport (254). It has been shown that external Cl
is necessary for sperm capacitation and fertilization (117, Although sperm chemotaxis is well established in marine
555). Considering that [Cl]i increases during capacitation animals (see sect. VI), its relevance in mammalian sperma-
(249), it has been hypothesized that its influx is coupled to tozoa has been a matter of debate for a long time (284).
the activity of a Cl/HCO3 antiporter. HCO3 influx Follicular fluid was found to attract capacitated human
through this exchanger would stimulate sAC and increase spermatozoa (95, 428, 533). The relatively low number of
the cAMP levels (555). responding cells (10% or less) complicates the interpreta-
tion of the results. Mammalian spermatozoa have been
Why is the capacitation-associated hyperpolarization needed shown to express olfactory receptors (401), although their
in spermatozoa? Initially it was proposed that the hyperpo- physiological function is not fully understood. One of these
larization was necessary to remove inactivation from CaV human olfactory receptors, hOR17 4, was detected in hu-
channels so they would be ready to open upon ZP stimula- man testicular cDNA, and bourgeonal, its agonist, is a che-
tion to achieve the AR (12, 15, 324). As we have discussed moattractant for human spermatozoa (477). Accordingly,
earlier, the functional involvement of CaV channels in ma- bourgeonal elevates human sperm [Ca2]i, and extracellu-
ture spermatozoa is now being debated (see sect. III). Alter- lar Ca2 is indispensable for both chemotaxis and the
natively, this hyperpolarization may be required to activate [Ca2]i increase. Pharmacological evidence suggests that
sNHE and increase pHi, which would in turn stimulate AC, but not PLC, is involved in the signaling cascade of
SLO3 and CatSper channels (381), and possibly sAC (541). bourgeonal (477). All membrane forms of ACs (mAC19)
A hyperpolarization would also increase the driving force have been detected in human and mouse spermatozoa (39,
for Ca2 influx through other Ca2-permeable channels 478). Furthermore, SQ22536 (ATP competitor of mAC)
blocks the bourgeonal-induced [Ca2]i increase and the hu-
such as TRPs, some of which are also stimulated by alka-
man sperm motility responses (chemotaxis and an increase
linization.
in flagellum beat frequency). These results are consistent
with the involvement of the cAMP cascade in bourgeonal
An increase in [Ca2]i seems to be important for capacita-
signaling in human spermatozoa. Ca2 imaging in immobi-
tion (FIG. 6), a matter that deserves further study. This in-
lized human spermatozoa revealed that the bourgeonal re-
crease could result from 1) a fast stimulation of CatSper sponse initiates in the flagellar midpiece and travels to the
(566) and/or CaV3 channels caused by the exposure of sper- head in seconds. Human spermatozoa display turns accom-
matozoa to albumin (163). Serum albumin induces an im- panied by asymmetric flagellar beating while swimming in
mediate increase in the CaV3 currents of spermatogenic the descending bourgeonal gradient, which resembles sea
cells, and increases their Ca2 window current by shifting urchin sperm chemotaxis (478) (see sect. VI). The Ca2
the voltage dependence of both steady-state activation and channels responsible for the Ca2 influx induced by bour-
inactivation (163). The increase could also result from 2) a geonal have not been determined, and the physiological
slower stimulation of CatSper and other pHi-sensitive chan- relevance of bourgeonal is a matter of debate (22, 284). It is
nels caused by the capacitation-associated alkalinization a perfume odorant apparently not present in the female
and 3) inhibition of the Ca2-ATPase and/or the Na/Ca2 reproductive tract. Therefore, bourgeonal may mimic the
exchanger resulting from cholesterol removal and/or in- function of an unidentified natural agonist. Bourgeonal can
creases in cAMP (190). attract 60% of the sperm population (478), while in an-

other report, human follicular fluid attracts only 10% (95), V. MAMMALIAN ACROSOME REACTION
suggesting that the signaling mechanisms are different
(284). Alternatively, if both chemoattracts share the same
mechanism, the natural agonist concentration in the follic- A. Generalities
ular fluid is very low.
As illustrated in FIGURE 1, the acrosome, a single mem-
In mouse spermatozoa, lyral, another perfume odorant, brane-delimited specialized organelle, overlies the nucleus
was also reported to function as a chemoattractant (187). In of many sperm species. The outer acrosomal membrane and
WT animals, 30% of the spermatozoa responded to lyral by the overlying plasma membrane fuse and vesiculate when
increasing the [Ca2]i. This is consistent with the observa- spermatozoa are presented with physiological inducers
tion that 30% of the spermatids express the lyral receptor from the female gamete, its vicinity, or to appropriate phar-
(MOR23). Spermatozoa from transgenic mice which over- macological stimuli. This unique, tightly regulated, irre-
expressed MOR23 respond better (up to 50%) to lyral than versible, single exocytotic process releases the acrosomal
those of WT. As the lyral concentration used in the sperm contents, modifies membrane components, and exposes the
chemotaxis assay was high (50 mM in capillary), distin- inner acrosomal membrane to the extracellular medium
guishing between real chemotaxis or trapping due to lyral (130, 131, 571). Sperm egg-coat penetration and fusion

toxicity must be considered. with the eggs plasma membrane require the release and
exposure of cell components resulting from this exocytotic
2. Thermotaxis process, named the acrosome reaction (AR). The egg enve-
lope, an important element of the gamete recognition and
The temperature of the female reproductive tract varies de-
signaling process, is also a protective layer (146, 549).
pending on the region and the sexual cycle (267). It has been
speculated that these intriguing temperature differences
External and internal Ca2 are essential for AR (130, 571),
could be caused by local variations in macromolecules, ir-
and a group of ion channel antagonists that block its flow
rigation microfluidics, and morphology (25, 268). The dis-
also inhibit this process. These findings emphasize the pre-
tal isthmus temperature is significantly lower than that of
ponderant role certain Ca2-permeable channels play in
the ampulla region in the pig (268). Furthermore, before
AR (134, 178, 418). As in other secretory cell types, AR
ovulation the temperature in the lower isthmus of rabbits,
requires soluble N-ethylmaleimide-sensitive attachment
the site of the sperm reservoir, is 0.8C lower than that in
protein receptors (SNAREs). The presence of sperm
the isthmic-ampullary junction, the site of the fertilization.
Temperature differences between two regions can be up to SNAREs has been documented in sea urchins (461, 462)
1.6C after ovulation, mainly due to a temperature decrease and mammals (reviewed in Ref. 352). Significant advance-
in the sperm reservoir (25). Rabbit and human spermatozoa ment in our comprehension of how the fusion machinery
display thermotaxis, that is, they preferentially swim to- operates during the sperm AR has been made utilizing per-
ward the warmer site (26). Studies on human sperm ther- meabilized human spermatozoa. These studies propose a
motaxis recently found unexpectedly that external Ca2 is working model for regulated sperm acrosomal exocytosis.
not essential for thermotaxis (24). In agreement with this In resting spermatozoa, SNAREs are assembled in inactive
result, blockers for many types of Ca2 channels did not complexes in the same membrane (cis-complexes). Sperm
inhibit thermotaxis, namely, Ni2 and nifedipine (CaV), AR stimulation triggers external Ca2 uptake leading to
ruthenium red (TRPV1), BCTC (TRPM8), and Gd3 RAB3A activation and SNARE complex disassembly. Free
(SOCs). In contrast, PLC-dependent intracellular Ca2 SNAREs are then able to reassemble in trans-complexes
mobilization seems important for thermotaxis, since where plasma membrane SNAREs interact with SNAREs in
BAPTA-AM (intracellular Ca2 chelator), U73122 (PLC the outer acrosomal membrane. This process may be facil-
inhibitor), and 2-APB (IP3R antagonist) inhibited the itated by synaptotagmin and complexin. At this stage, the
process. Recently, we identified TRPM8 channels in hu- trans-complexes are arrested and resistant to tetanus toxin
man spermatozoa and confirmed their functionality but sensitive to botulinum neurotoxin B, until a local Ca2
through recording [Ca2]i increases induced by menthol increase coming from the acrosome through IP3Rs activates
and cooling, which were inhibited by specific TRPM8 the synaptotagmin-dependent relief of the complexin block,
antagonists BCTC and capsazepine (143). Although the and acrosomal exocytosis is completed (86, 352).
TRPM8 temperature threshold for activation is lower
than the temperature of the female genital tract, many The acrosome functions as a Ca2 store whose contents are
parameters such as lipid compositions, particularly phos- released during AR. The molecular identity of the physio-
phoinositides (439), phosphorylation state (1), and the logical AR inductor(s); when, where, and how this reaction
[Ca2]i (439) can modify this threshold. Considering that takes place; and the main actors participating in this indis-
the temperature of the sperm reservoir decreases after pensable process have been the subject of intense research
ovulation, TRPM8 channels could alternatively modu- for years, and a current matter of lively debate (i.e., Ref.
late sperm motility (i.e., hyperactivation). 191).

DARSZON ET AL.
The classical view, although presently under scrutiny, pro- vation (490), induce chemotaxis (158), and finally poten-
poses that the zona pellucida (ZP), the extracellular coat tiate the ZP-induced AR (440) and trigger AR itself (32).
surrounding the egg, is the physiological inductor of the The [Ca2]i rise and AR induced by progesterone are
mammalian sperm AR. ZP is composed of a matrix of four insensitive to pertussis toxin (PTX), suggesting different
glycoproteins (ZP1-ZP4), except for mouse ZP that con- signaling mechanisms to those involved in the ZP-
tains only three glycoproteins (367). The importance and triggered AR (30, 375, 504).
site of ZP protein glycosylation is also under revision (191).
ZP3 is considered the main physiological inductor of the Soluble ZP and/or ZP3 initiate a set of responses in capac-
mammalian sperm AR (177, 550) though in the human itated spermatozoa that will lead to a sustained [Ca2]i
system ZP4 and ZP1 can induce this reaction to a lesser increase needed to achieve AR. Important among these is a
extent (76, 200, 282). In spite of these, recent experiments pHi elevation (179), apparently regulated by G proteins
in which oocytes expressing genetically modified mouse (11). Gi, Gq, and Gs have been reported to be present in
ZPs are used show that very few spermatozoa bound to ZP mammalian sperm (40, 539, 544). In mouse spermatozoa
have undergone AR after 1 h (28, 191). This puzzling ob- ZP can activate Gi (545) and PTX, a specific inactivator of
servation reopens questions about the nature of the physi- Gi, inhibits the ZP-induced pHi change and the AR (15).
ological inducer of AR and the site at which this process PTX also inhibits AR and many of the ion fluxes associated

occurs. Examination of gamete interactions within the first with it in bovine and human spermatozoa (179, 315). These
minutes under physiological conditions using oocytes ex- results indicate the importance of the pHi change induced
pressing genetically modified ZPs and labeled spermatozoa by ZP for the AR. In addition, this process involves Em
to detect AR are required to answer these fundamental changes (15) that require further investigation.
questions. It is fortunate that genetically modified mouse
spermatozoa that express GFP in their acrosome and red Also associated with AR triggered by ZP and other induc-
fluorescent protein in their mitochondria are now available. tors are increases in cAMP, protein phosphorylation, and
These spermatozoa fertilize normally in vivo and in vitro phospholipid turnover (69, 178, 190, 441). The presence
and activation of PLCs, PKC, and PKA during AR have
and can be observed exciting with light through the dis-
been documented (30, 68, 185). As anticipated, the ZP and
sected uterine and oviductal walls as they migrate through
progesterone-induced Ca2 permeability changes associ-
the female reproductive tract, as well as during AR (244).
ated with AR are also influenced by PKC and PKA inhibi-
On the other hand, as gamete interaction and the AR are
tors in mammalian spermatozoa (67, 178, 180, 243). As
species specific, it is believed spermatozoa possess special-
described in the capacitation section (sect. IVF), protein
ized receptors for ZP (417). Although several ZP3 receptor
tyrosine kinases are present in mammalian spermatozoa,
candidates have been proposed (159), their physiological
and their antagonists can inhibit ZP (317, 420), progester-
relevance is still unclear, since knockout mice for these can-
one (294, 338, 354, 429, 504), and thapsigargin-induced
didates are fertile or subfertile (21, 470, 550). Evidence is
AR (156). Tyrosine kinases such as Src (detected in bovine
accumulating that gamete interaction may involve multiple
spermatozoa) were shown to participate together with PKA
recognition sites (389, 527). and ouabain (a specific Na-K-ATPase inhibitor present
in the female tract) in the transactivation of EGF receptor
At the present time, Izumo is the only sperm protein known (EGFR) leading to [Ca2]i increases and the AR (133). Ty-
to be necessary for sperm-egg interaction. It is a member of rosine kinases and phosphatases have indeed been proposed
the immunoglobulin superfamily; knocking it out results in as key elements for AR. Findings indicate that dephosphor-
sterile mice whose spermatozoa are not able to fuse with the ylation of synaptotagmin by calcineurin must occur at a
egg plasma membrane (273). It is not known how Izumo particular time during this process in human spermatozoa
intervenes in the fusion process. Recently it was reported to achieve exocytosis (86). It is worth noting that syntaxin,
that Izumo relocalizes from the anterior acrosome where it synaptotagmin, and SNAP25 can associate with CaV2.1
is found in intact mouse spermatozoa, to other regions in and CaV2.2 channels. Therefore, both types of channels
the head engaged in fusion with the egg plasma membrane. have been found as components of the exocytotic vesicle
Tssk6, a member of the testis-specific serine kinase family, is docking/fusion machinery (17, 89). Further studies are re-
necessary for the movement of Izumo, and its role is medi- quired to fully understand the interrelationship between
ated by the actin cytoskeleton (362, 476). [Ca2]i and kinase/phosphatase activity during intermedi-
ate steps of the physiologically relevant AR that involve the
Other cellular ligands can induce the AR such as proges- fusion machinery.
terone (31, 55, 355, 506), -aminobutyric acid (GABA)
(280, 355, 559), glycine (301), EGF (133), ATP (339,
442), acetylcholine (66), and sphingosine-1-phosphate B. Ca2 and the Acrosome Reaction
(495). The role of many of these alternative agonists is
not clear. For example, progesterone has been proposed A common and fundamental feature of physiological and
to facilitate capacitation (35), promote sperm hyperacti- pharmacological AR inducers is that they provoke intracel-

lular multicomponent Ca2 rises. At least three separate yet CaV3 channels, including those in spermatogenic cells and
linked Ca2 channels have been proposed to participate in testicular sperm, inactivate at potentials more positive than
these responses. Triggering of the sperm AR involves sig- 60 mV (11, 137, 407). Therefore, in cells such as nonca-
naling bifurcations that regulate G protein activation, pacitated mouse spermatozoa, in which the resting poten-
cAMP, and pHi. The complexity of the [Ca2]i changes that tial is more positive than 60 mV, removal of CaV3 inac-
trigger AR results from different levels of integration of the tivation requires hyperpolarization of Em. Taking this into
signaling events regulating these fluxes. Molecular identifi- account, it was proposed that the capacitation-associated
cation of the channels involved in this process has been the hyperpolarization in mouse spermatozoa (see sect. IVF) is
subject of study of several research groups using diverse important to render CaV3 channels ready to open during
experimental approaches (105, 134, 178, 433). AR (12, 138). Still, once inactivation is removed, CaV3
channel opening requires a depolarization. The section on
The proposal that CaV channels participate in AR evolved capacitation presents a discussion of possible alternative
from functional and pharmacological observations (re- purposes of the hyperpolarization that occurs in mouse
viewed in Refs. 136, 178) (TABLES 1 and 2). For example, spermatozoa during this process.
the spermatozoa of many mammalian species undergo
[Ca2]i increases sensitive to CaV channel blockers when If CaV3 channels participate in AR, how does ZP induce a

depolarized by elevating [K]e. These changes depend on depolarization in capacitated mouse spermatozoa to open
external pH and Ca2 and can lead to AR (15, 23, 63, 176, them? A nonselective, cation channel could mediate this
327). depolarization (15). Poorly selective mammalian sperm cat-
ion channels registered in planar bilayers (111, 305) and in
C. Initial Transient Ca2 Rise patch-clamp studies (162) are candidates for causing this
depolarization. Additionally, as mouse and human sperma-
tozoa have a Cl equilibrium potential of approximately
When exposed to solubilized ZP3, individual mouse sper-
30 mV (209, 248), they would depolarize upon Cl chan-
matozoa undergo an initial fast (200 ms) and transient
nel opening. These cells possess the niflumic acid-sensitive
[Ca2]i elevation compatible with the kinetic and pharma-
anion channels documented in patch-clamp studies (162).
cological properties of CaV3 channels. Thereafter, a signif-
Furthermore, ionotropic GABA and glycine receptors have
icantly slower and sustained [Ca2]i increase occurs lasting
been detected in mammalian spermatozoa, and their li-
up to minutes (12). As mentioned earlier, patch-clamp stud-
gands can induce the AR. Strychnine, an antagonist of the
ies in mouse spermatogenic cells revealed mainly CaV3 cur-
glycine receptor, inhibits the AR induced by recombinant
rents, and all isoforms were reported to be present (11, 324)
(TABLE 1). To distinguish the CaV3 family member respon-
human ZP3 (65), and a null mutant of the glycine receptor
sible for these currents, two of three isoforms were knocked is subfertile and cannot undergo ZP-induced AR (453).
out. CaV3.1 knock-out mice are fertile, and their spermato- These findings suggest that the opening of Cl channels
genic cells display CaV3 currents similar to those of WT could depolarize capacitated spermatozoa to open CaV
animals (479), suggesting that CaV3.2 channels are respon- channels and trigger AR, a possibility worth further explo-
sible for the currents. This finding is consistent with the ration.
2
Ni blocking profile of the CaV3 currents in spermatogenic
cells and their location in the head area of spermatozoa Altogether, molecular and pharmacological evidence sup-
(514). Surprisingly, CaV3.2 null male mice are also fertile ports the role of CaV channels during AR, particularly in
even though their spermatogenic cells basically do not dis- mouse spermatozoa (TABLES 1 and 2). However, although
play CaV3 currents (114, 160, 479). To discard compensa- now it is possible to perform patch-clamp recordings in
tory processes, it will be necessary to use the double testicular and epididymal mouse spermatozoa as well as in
(CaV3.1, CaV3.2) knockout mice. ejaculated human spermatozoa, CaV currents have only
been detected in testicular sperm, raising the question as to
CaV3.3 has not been knocked out; however, the slower whether these channels are functional in epididymal and
kinetics of its currents (406) makes it a less likely candidate ejaculated sperm. It is surprising that of all the ion channels
to produce the CaV3 currents observed in mouse spermato- whose message and proteins described as being present in
genic cells. In addition, CaV3.3 channels immunolocalized mouse spermatozoa, only CatSper and SLO3 currents, pres-
to the flagella, whereas CaV3.1 and CaV3.2 are found at the ent in the principal piece, have been detected by patch
sperm head, the site of AR (514). Additionally, CaV3 cur- clamping the cytoplasmic droplet of epididymal spermato-
rents in spermatogenic cells display sensitivity to com- zoa. Furthermore, from all mice lacking ion channels, only
pounds such as urocortin, fenvalerate, and gossypol that mice null for CatSper and Slo3 are infertile. However, it is
may explain their contraceptive properties (27, 502, 567). It reasonable to speculate whether, under these conditions,
is noteworthy that none of the individual CaV channels that there are modes of regulation in spermatozoa that function-
have been knocked out generated an infertile mouse pheno- ally silence the activity of these channels. As discussed, CaV
type (114, 291, 445) (see TABLE 1). currents disappear from testicular to epididymal spermato-

DARSZON ET AL.
zoa, and in addition, neither CNG channels nor Kirs have an intertwined network of signaling cascades (233, 327,
been detected in the latter. Is this due to potential technical 418, 505, 512). Single-cell Ca2 imaging experiments show
problems, such as difficulties in clamping the voltage that almost all cells respond to progesterone with a biphasic
throughout this morphological complex cell and/or a rela- Ca2 increase; however, only 20% undergo AR, indicat-
tive scarcity of these channels? It must be stressed that ca- ing that other mechanisms must be activated concomitantly
pacitation involves membrane reorganization and a com- to achieve exocytosis (32). For instance, Src inhibitors de-
plex set of phosphorylation changes that might unmask the crease the Ca2 rise and the AR induced by progesterone
activity of CaV and other channels in preparation for a role (528). The proposal is that Src is activated by progesterone
during AR, whereas novel modes of CaV3 channel regula- upstream of the Ca2 increase and that it may be involved in
tion (see sect. IIIA) as well as possible CaV3 channel inter- Ca2 release from intracellular stores, such as the RNE,
actions with as yet undefined partners may inhibit the ac- considering its postacrosomal localization. These results
tivity of these channels during patch-clamp experiments in are consistent with the inhibition of the progesterone-in-
epididymal spermatozoa prior to capacitation. For induced [Ca2]i plateau phase caused by general tyrosine ki-
stance, as indicated earlier 6, a CaV auxiliary subunit, in- nase antagonists (59). Src is known to regulate [Ca2]i in
hibits CaV3 channels (116). Although it is not known if this different cell types either by affecting Ca2 release from
subunit is present in spermatozoa, it is tempting to speculate intracellular stores or by phosphorylation of CaV channels

that inhibition by 6 could maintain CaV3 channels silent in (528).
epididymal spermatozoa, and its removal during capacita-
tion would reactivate them.
D. Sustained Ca2 Rise
In spite of these arguments, the observation that spermato-
zoa from CaV3.2 null mice basically display no residual The initial and transient Ca2 elevation triggered by solu-
CaV3 currents, yet remain fertile, is difficult to reconcile bilized ZP or ZP3 is followed by a slow and prolonged Ca2
with their participation in AR. Unless spermatogenesis is elevation that can last several minutes. Since the early days
particularly rich and versatile in its ability to compensate of studying sperm Ca2 transport, it was known that a high
for missing CaV channels and for their expression time, the and sustained Ca2 influx is necessary for the AR to occur
role of these channels in fertilization is open to question. In (11, 15, 179, 306, 454, 456, 571). How is this second Ca2
spite of this, there is evidence suggesting that CaV3 channels elevation generated? The current view is that the initial and
may participate in sea urchin sperm chemotaxis (284, 563). transient Ca2 rise activates a Ca2-sensitive PLC- (185)
that hydrolyzes PIP2 to produce DAG and IP3 (539). The
CaV channels are also present in human spermatozoa, but fact that PLC-4 null mice are infertile and unable to un-
their role during AR is even more controversial, due in part dergo the ZP3-induced AR supports this model (185). In-
to the lack of native ZP for experimentation (reviewed in deed, it has been shown that ZP/ZP3 induces IP3 increases
Ref. 134). There is indirect evidence of their activity in in mouse spermatozoa (510). Binding of IP3 to its receptor
human spermatozoa (47, 56, 276, 400, 418, 466). Fluores- (located in the acrosome and the RNE, FIG. 1 and TABLE 3)
cence determinations simultaneously measuring [Ca2]i releases Ca2 from one or both stores (142, 251, 260, 391,
and Em showed that the addition of K in the presence of 539), which in turn, as in somatic cells, would induce the
valinomycin depolarizes human sperm and induces [Ca2]i opening of Ca2 channels at the plasma membrane (SOC
increases; however, these [Ca2]i increases were insensitive activity). As mentioned before, several TRPCs are present in
to classical CaV blockers (nifedipine and verapamil) but spermatozoa, and in particular TRPC2 has been implicated
sensitive to Ni2 (327). in mouse AR (283). Considering that TRPC2 null mice are
fertile (484), that human TRPC2 is a pseudogene (575), and
Recently, recombinant proteins as well as native ZP isolated that the molecular composition of the SOC machinery
from human oocytes recovered after in vitro fertilization seems to be more complex than previously thought, it is
procedures have helped to define better the participation of likely that other components such as STIM, ORAI, and
CaV channels in AR. Pharmacological studies measuring other TRPCs may be also involved in the sustained Ca2
AR induced with either native ZP or recombinant ZP3 also entrance. This contention is supported by the reported pres-
suggest the presence and participation of CaV channels dur- ence of STIM1 in human sperm (105) (A. Snchez-Tusie,
ing human AR (consult TABLE 2 and references therein). unpublished results).
Progesterone at concentrations found in female genital flu- Additionally, the possible regulation of the Ca2 channels
ids (M) influence several aspects of sperm behavior (see responsible for the sustained increase in [Ca2]i needed for
sect. IVE), AR among them (reviewed in Ref. 32). The na- AR by proteins like homer (350), enkurin (498), junctate
ture of the channels involved in the progesterone-induced (480), PKDREJ (497) etc., and mechanisms that affect their
increase in [Ca2]i is not clear, although it may involve a activity (PIP2 levels, phosphorylation, pH, etc.) clearly re-
combination of CaVs, SOCs, and intracellular channels in quire more attention.

FIGURE 7 summarizes a working signaling model of the flux. After IP3 production by PLC-, Ca2 release from
components and events leading to the mammalian sperm IP3-sensitive intracellular stores would activate SOCs re-
AR. Solubilized ZP3 induces a fast and transient increase in sponsible for the sustained [Ca2]i elevation. The figure
[Ca2]i that could involve CaV channels, although other also summarizes our view of parallel and concomitant steps
Ca2-permeable channels may be responsible for this in- necessary to achieve AR that occur thereafter. This signal-

DARSZON ET AL.
ing cascade involves preparation of the fusion machin- solubilized ZP mediated by CatSper1 is not essential for AR
ery that includes SNAREs, -SNAP/NSF, synaptotagmin, (433). Why then is AR inhibited by CaV3 channel blockers?
Rab3A, and recently incorporated, exchange protein di- It would be informative to test CaV blockers (i.e., nimodip-
rectly activated by cAMP (Epac) (64), a GEF protein that ine, mibrefadil, etc.) to corroborate the pharmacological
links cAMP and Ca2 signaling (reviewed in Ref. 352). It profile of the AR and the participation of CaV channels in
has been proposed that a sAC is activated by Ca2 during CatSper null spermatozoa.
the sustained ZP3-induced Ca2 rise, that the resulting
cAMP activates Epac and that the signaling cascade It is also notable that spermatozoa from CatSper null mice
branches in at least two arms that come back together at the undergo the normal tyrosine phosphorylation associated
end of the cascade. Dissecting the roles of sAC and mACs with capacitation and can fertilize zona-free eggs (83, 426,
during AR requires further work. One branch prepares the 432, 565). Furthermore, the [Ca2]i rise induced by solubi-
tethering of the SNAREs fusion machinery involving lized ZP (or progesterone and thapsigargin; Ref. 355) has
Rab3A activation, and the other one activates PLC- via been reported to commence in the mammalian sperm head
Rap stimulation, to again produce IP3 and release Ca2 (175, 185, 186, 468) and not in the principal piece as re-
from internal stores, the final trigger for membrane fusion ported by Ren and Xia (433). The correlation with high
(64). The existence and regulation of two waves of IP3 spatiotemporal resolution [Ca2]i changes and AR in single

production and Ca2 release from internal stores require spermatozoa is important to understand this fundamental
further investigation. process required for fertilization. Now it is possible to im-
age the acrosome status using a transgenic mouse which
expresses GFP in the acrosome (379) or fluorescence probes
E. CatSper Channels and the (186, 240, 509).
Acrosome Reaction
CaV3 currents have not been recorded from mouse epidid- VI. SPERM CHEMOTAXIS IN MARINE
ymal spermatozoa (433), and both the CaV3.1 and CaV3.2 ANIMALS
null mice are fertile (122, 479). These findings have led to
the consideration of the participation of CatSper channels In contrast to terrestrial animals, most marine animals re-
in the solubilized ZP induced elevation of [Ca2]i (566). lease their gametes into the ocean where unlimited dilution
Ren and Xia (433) reported that solubilized ZP initiated an will increase the distance separating them. In these species,
early [Ca2]i elevation, mostly within 20 s, in the principal successful fertilization against all odds requires efficient
piece of the sperm flagella, where CatSper proteins are pres- sperm chemotaxis towards the egg. To improve the proba-
ent, that propagated to the sperm head within a few sec- bility of gamete interaction, female gametes of many marine
onds. Around 35% of the cells exposed to ZP displayed an animals have evolved to exploit diffusible molecules that
additional delayed response separated from the early one. activate and attract their homologous spermatozoa (360).
Spermatozoa from CatSper1 null mice lose the early [Ca2]i Sperm chemotaxis is a fascinating biological phenomenon
increase (within 2 min) induced by solubilized ZP. This in which spermatozoa rapidly and precisely recognize a che-
response was rescued by spermatozoa from mice expressing moattractant gradient and regulate their swimming direc-
a functional GFP tagged CatSper1. These results indicate tion toward the source of the chemoattractant (227). It is
that solubilized ZP activates CatSper channels provoking known that Ca2 plays fundamental roles in this process
the early [Ca2]i increase. It is worth noting that the time without exception (284).
resolution employed in this work (2 frame/s) would not
allow reliable detection of the fast ZP-induced opening of
CaV3 channels (100 ms) reported (11). Interestingly, sper- A. Sea Urchin Spermatozoa
matozoa from CatSper null mice still undergo the solubi-
lized ZP-induced AR as well as those from WT mice, and Although the first molecular identification of a sperm che-
20% of them display the delayed [Ca2]i elevation. These moattractant was achieved in the 1880s in the plant king-
findings suggest that the early [Ca2]i increase induced by dom (bracken ferns) (410), it is in sea urchins where our
FIGURE 7. Calcium mobilization elicited upon ZP3 binding to sperm receptor(s) during the mammalian acrosome reaction. ZP3 binding
activates a Gi protein likely involved in the pHi increase, and may also activate CaV channels inducing a membrane depolarization (caused by GABA
or an unidentified cation channel). This initial and transient Ca2 rise stimulates PLC- that catabolizes PIP2 to produce IP3 and DAG. IP3 binds
to its receptor located in the acrosome and/or in the RNE, liberating stored Ca2 and activating SOCs (TRPCs and/or ORAI), possibly through
STIM aggregation. DAG and PIP2 may also stimulate TRPCs channels. The resulting sustained Ca2 increase activates sAC, which elevates
cAMP turning EPAC on. EPAC signaling branches in two arms: 1) activation of Rab3A, triggering the tethering of the acrosomal and plasma
membranes through the stimulation of RIM and SNAP/NSF to render the SNARE machinery ready for fusion, and 2) activation of Rap, which
in turn stimulates PLC- to generate IP3 and liberate Ca2, the final trigger for membrane fusion. Low and high Ca2 levels are maintained in
the cytosol (PMCA) and Ca2 stores (SPCA1 and SERCA) by ATP-driven Ca2 pumps.

understanding of the mechanisms underlying sperm che- gated K-selective channel (tetraKCNG/CNGK channel)
motaxis has advanced furthest (98, 135, 227, 284). Sea (60, 194, 195). The tetraKCNG/CNGK channel is a new
urchin eggs are covered with an extracellular matrix, called type of K channel composed of a single polypeptide that
egg jelly (EJ), mainly composed of polysaccharides, which contains 24 TM segments (4 subunits of 6 TM segments).
function as species-specific ligands to induce the AR (see Recently, it was demonstrated that the KCNG channel of A.
sect. VII). The EJ also contains small diffusible peptides, punctulata spermatozoa is activated when cGMP binds to
which activate sperm motility in slightly acidified seawa- the third cyclic nucleotide binding domain (CNBD), with
ter (392). These peptides, collectively named sperm-acti- high affinity (K1/2: 26 nM for cGMP and 17 M for cAMP)
vating peptides (SAPs) (499), induce rapid physiological (60). The tetra-KCNG/CNGK channel does not have an
changes in sperm cyclic nucleotide levels, Em, pHi, and intrinsic inactivation mechanism; therefore, its activation
[Ca2]i (135, 284). Sperm chemotaxis in response to a will depend on cGMP levels. The SAP-induced hyperpolar-
ligand was first documented in the animal kingdom using ization affects several important Em-dependent membrane
the purified ligand resact (CVTGAPGCVGGGRL-NH2), proteins such as CaV channels (removal of inactivation)
a SAP derived from Arbacia punctulata (546). Con- (223, 485), hyperpolarization-activated cyclic nucleotide-
versely, speract (GFDLNGGGVG) was the first purified gated (HCN) channels (Na influx) (197, 211), Na/H
and structurally identified SAP from Hemicentrotus pul- exchangers (NHE) (pHi increase) (437), and K-dependent

cherrimus (499) and Strongylocentrotus purpuratus Na/Ca2 exchangers (NCKX) (Ca2 extrusion) (487). El-
(237). evation of pHi together with the hyperpolarization activates
AC and increases cAMP level (45), which potentiates Na
As a consequence of intensive efforts in time-resolved fluo- influx (211). The pHi increase also inactivates the guanylyl
rometry (286, 351, 387, 388, 485), Ca2 imaging (58, 227, cyclase through dephosphorylation of its catalytic domain
561563), and molecular identification (60, 195, 211, 223, (547) and enhances the activity of phosphodiesterase-5
486, 487), our knowledge about the signaling cascade trig- (486), which is activated by cGMP itself (444) and also by
gered by SAPs has advanced significantly in the last 10 phosphorylation mediated by PKA upon EJ treatment
years. FIGURE 8 depicts a proposed general scheme for SAP (486).
signaling. SAPs may bind directly to guanylyl cyclase [i.e.,
resact (471) and asterosap (351)] or to its associated recep- After hyperpolarizing, Em is displaced towards the resting
tor [i.e., speract (132)] in the flagellar plasma membrane level as HCN channels open and possibly tetra-KCNG/
stimulating the synthesis of cGMP (286). The elevation of CNGK channels close. This Em change initially activates
cGMP induces K efflux by activating a cyclic nucleotide- CaV3 channels and then possibly CaV1 and CaV2 channels.
FIGURE 8. Signaling events regulating [Ca2]i and sea urchin sperm swimming. A: after binding to its receptor, speract stimulates a
membrane guanylate cyclase (GC) and elevates cGMP that activates tetrameric cGMP-regulated K channels (tetraKCNG) leading to membrane
potential (Em) hyperpolarization. This Em change stimulates hyperpolarization-activated and cyclic nucleotide-gated channels (HCN), removes
inactivation from voltage-activated Ca2 channels (Cav), facilitates Ca2 extrusion by K-dependent Na/Ca2 exchangers (NCKX), and activates
Na/H exchangers (NHE) and adenylate cyclase (AC) activity. HCN opening and Na influx contribute to Em depolarization, and increases in
[Ca2]i and Na further depolarize Em. Elevated [Ca2]i enhances flagellar bending, leading the spermatozoon to turn. Possibly, the [Ca2]i
increase also opens Ca2-regulated Cl channels (CaCC) and/or Ca2-regulated K channels (CaKC), which then contribute to hyperpolarize
again the Em, removing inactivation from Cav channels and opening HCN channels. This mechanism is then cyclically repeated to generate a train
of Ca2 increases that produce a sequence of turns. The sequence continues until one or more of the molecular components in the pathway are
downregulated. cAMP activates a poorly characterized Ca2 influx pathway, which may contribute to a tonic [Ca2]i increase. B: a spermatozoon
(green) swimming in a circular trajectory in a chemoattractant gradient (gray background) is periodically stimulated due to changes in the rate
of chemoattractant capture. Top panel: Chemotactic behavior of a single L. pictus sperm. When swimming in an ascending gradient, the onset
of [Ca2]i fluctuations is suppressed until the spermatozoon senses an ascending to descending gradient inversion (red circle). After a 200-ms
delay, the spermatozoon undergoes a [Ca2]i fluctuation just before reaching the gradient minima (white circles, fluctuations from many
experiments). Consequently, the spermatozoon executes a turn-and-run episode that brings it closer to the chemoattractant source (black
arrow). Bottom panel: nonchemotactic behavior of a single S. purpuratus sperm. The onset of [Ca2]i fluctuations is not suppressed by an
ascending chemoattractant gradient. They occur at random; therefore, the turn-and-run episodes promote sperm relocalization but not a
directed approach to the chemoattractant source. C: model of chemotaxis in sea urchin spermatozoa. A chemotactic spermatozoon swimming
in a chemoattractant gradient (background) undergoing cyclic changes in Em from resting to a hyperpolarized state (Hyp, green shadow) and then
to a depolarized state (Dep, blue shadow) that control Cav activity. The spermatozoon path is depicted as a black arrow as it swims in a
chemoattractant gradient (background). The incremental activation of speract receptors in spermatozoa experiencing an ascending gradient
leads to extended hyperpolarization which suppresses Ca2 fluctuations (i). The hyperpolarization reverses once sperm enter a negative speract
gradient, which after 200-ms delay (red line) generates a chemotactic turn that reorients the spermatozoon towards the gradient source (ii).
The onset of each [Ca2]i fluctuation is denoted by the black circles. Repolarization due to GC inactivation, decrease in cGMP levels by
degradation, Na influx through HCN channels, and other unknown depolarizing elements open Cav channels, possibly of the T and L type (ii). The
black (low) to red (high) pseudo-color shade around the trajectory illustrates the [Ca2]i changes in the flagella. Note that straight swimming
coincides with elevated [Ca2]i. At some point during the straighter swimming phase in the positive speract gradient, a hyperpolarized Em is
reestablished and extended by continuous speract receptor recruitment (iii), which once again reverts to depolarized Em as sperm leave the
positive gradient (iv). This sets up a sequence of chemotactic turns, triggered by hyperpolarization/depolarization cycles that serve as the
primary translators of the state of the extracellular chemoattractant gradient.

DARSZON ET AL.
Transcripts for CaV3.3, CaV1.2, and CaV2.3 were obtained [Ca2]i increase (561), explaining the previous observa-
from S. purpuratus testis (223, 485) (TABLE. 1). Immuno- tions. Time-resolved fluorometry using stopped-flow or
staining indicated that CaV1.2 channels are localized on the caged compounds showed a significant time delay (150
sperm flagellum (223) (TABLE 1). Initially CaV channels 600 ms) for the [Ca2]i increase after spermatozoa were
were not considered important for SAP-induced Ca2 stimulated by SAPs or caged cGMP (286, 351, 387, 388,
changes, mainly because blocking them did not inhibit the 503). This time lag well corresponds to the time required to
speract-induced Ca2 increase measured in sperm popula- achieve the hyperpolarization induced by SAPs or cGMP
tions by fluorometry (455). Thereafter, Ca2 imaging of (485) that removes inactivation followed by activation of
individual immobilized S. purpuratus spermatozoa revealed the CaV3 channels. This delay for the [Ca2]i increase must
that speract induced rapid [Ca2]i fluctuations superim- be fundamental for the mechanism of sperm chemotaxis as
posed with a tonic [Ca2]i increase originating from the described later. It is reasonable to consider that the [Ca2]i
flagellum, both being dependent on external Ca2 (561). fluctuations induced by speract (561) are caused by regu-
CaV channel blockers, such as Ni2 and nimodipine, inhib- lated hyperpolarization-depolarization Em cycles. Techni-
ited only the sperm [Ca2]i fluctuations, but not the tonic cally challenging single sea urchin sperm Em measurements

are needed to corroborate this explanation. The current when swimming in descending speract gradients, close to
hypothesis is that Ca2-dependent Cl and K channels where the gradient changes polarity, while S. purpuratus
might be involved in the rehyperpolarization of the sperm spermatozoa generate Ca2 fluctuations and turn without
Em after Ca2 influx through CaV channels. These ele- distinguishing the direction of the chemoattractant gradi-
ments could generate Em oscillations without requiring ent. This different behavior emanates from the ability of L.
cGMP fluctuations, which anyway are likely to occur by pictus spermatozoa to selectively suppress their Ca2 fluc-
repetitive stimuli with SAPs during sperm chemotaxis tuations when swimming toward the source of the chemoat-
under experimental conditions where spermatozoa swim tractant. A central feature of sea urchin sperm chemotaxis,
in two dimensions in circles close to the surface of the and possibly of sperm chemotaxis in general, is tuning of
coverslip (58, 227). the Ca2 fluctuations and the associated turning episodes in
the direction of the chemoattractant gradient. The physio-
Although the mechanism responsible for the tonic Ca2 logical significance of the distinct behavior of spermatozoa
influx is unknown, it has been proposed that cAMP regu- from these two sea urchin species may stem from differences
lates it (97, 387). As all CatSper-related genes were found in in their respective habitats such as varying hydrodynamic
the sea urchin genome (see sect. IVD), this channel, which is environments and/or in their reproductive cycles, animal
activated by the pHi and cAMP increases induced by spe- density, and gamete spawning synchronization (227).

ract, is a good candidate. Further experiments are required
to advance our understanding of the SAP signaling cascade. So far chemotaxis has been mostly studied in spermatozoa
swimming in two dimensions, that is, spermatozoa under-
Time-resolved Ca2 imaging using caged cGMP revealed going thigmotaxis on the coverslip surfce (104). Under
that only the Ca2 transient sensitive to Ni2 and nimodip- physiological conditions, sea urchin spermatozoa swim in
ine induces asymmetric flagellar bending that results in a three dimensions describing helical trajectories with geo-
highly curved trajectory altering the sperm swimming direc- metric characteristics distinct from those they display in
tion; the remaining tonic [Ca2]i increase does not (562). two dimensions (102). Spermatozoa swim 25% slower in
Furthermore, detailed analysis of the relationship between two dimensions, and their circular trajectories have
flagellar [Ca2]i and the degree of its asymmetry revealed a 130% larger radius of curvature than those swimming in
three dimensions. These results point out the necessity of
positive correlation only during the very early phase of the
characterizing the chemotactic response of spermatozoa in
[Ca2]i increase, but not for the entire process (135, 563).
three dimensions.
In other words, after a short asymmetric period, the flagel-
lum bend returns to a less asymmetric form while the
As usual, novel findings generate new questions, and this is
[Ca2]i is still elevated. Therefore, the observations indicate
the case for sea urchin sperm chemotaxis. 1) Does sperm Em
that a proportional relationship between [Ca2]i and the
oscillate during the [Ca2]i fluctuations triggered by SAPs?
degree of flagellar asymmetry does not hold in intact sea
2) What is a molecular identity of the Ca2 channels that
urchin spermatozoa. What determines the return to less
contribute to the tonic Ca2 influx induced by SAPs?
asymmetric flagellar form despite continuing high [Ca2]i
3) What determines the symmetric flagellar form at high
levels is a new and open question. [Ca2]i during sperm chemotaxis? 4) What is the molecular
explanation for the behavioral difference between S. purpu-
In spite of the fact that sea urchin SAPs induce very similar ratus and L. pictus? 5) How do spermatozoa respond to
physiological responses in spermatozoa from their corre- SAPs in three dimensions?
sponding species, chemotaxis had been demonstrated only
in A. punctulata spermatozoa towards resact (286, 546).
Although speract redirects the swimming paths of S. purpu- B. Sea Squirt (Ascidians) Spermatozoa
ratus spermatozoa with a stereotypical turn-and-run pat-
tern (FIG. 8), under laboratory conditions it has not been Spermatozoa of the sea squirt Ciona intestinalis display
shown to be a chemoattractant (reviewed in Ref. 135). Re- clear chemotaxis in response to gradients of a sulfated ste-
cently our group discovered that speract is chemotactic to- roid named SAAF (sperm-activating and attracting factor),
wards L. pictus spermatozoa (227). Notably, we now have which is released only from unfertilized mature oocytes
been able to characterize spermatozoa of two phylogeneti- (296, 576, 578). Since the SAAF receptor has not been
cally closely related sea urchins that react to speract gradi- identified, the signaling mechanism is not well character-
ents with turn-and-run type motility responses, yet only one ized. It is known that an Em hyperpolarization is an impor-
of these species is chemotactic. A detailed analysis was car- tant initial step (275) and the tetraKCNG/CNGK channel
ried out for individual sperm trajectories and flagellar has been identified in the C. intestinalis genome (60), sug-
[Ca2]i in response to a chemoattractant gradient generated gesting that the initial signaling steps might be similar to
by photoactivating caged speract (503). The most striking those of sea urchin spermatozoa. Also, the general sperm
difference between the two species was that L. pictus sper- behavior during chemotaxis is almost identical to that de-
matozoa selectively undergo Ca2 fluctuations and turn scribed in the sea urchin; that is, spermatozoa change swim-

DARSZON ET AL.
ming direction upon facing a descending SAAF gradient puratus (suPLC) sperm extracts with an anti-PLC2 anti-
which triggers a Ca2 spike that generates a chemotactic body against the mammalian isoform (106). Although
turn (465). The degree of flagellar asymmetry and the PLC- regulation is not well understood, PLC-1 (the most
[Ca2]i are positively correlated only at the initial phase similar to PLC-2) is regulated by Ca2, the GTP-binding
(465), as observed in sea urchin spermatozoa. However, proteins Rho and Gh, as well as by the levels of PIP2 and IP3
CaV blockers do not affect chemotaxis; instead, SKF96365, (430). In sea urchin spermatozoa, immunofluorescence and
a general TRP channel blocker (see sect. IIIC), inhibits che- immunogold experiments showed that Rho localizes in the
motaxis (577). Interestingly, the first enzyme containing a acrosomal region, the middle piece of the head, and in the
functional voltage sensor domain besides voltage-gated flagellum. Rho was also found in a preparation enriched in
channels, a voltage-sensitive phosphatase that hydrolyzes acrosomal and plasma membranes colocalizing (in a con-
plasma membrane PIP2, was discovered in C. intestinalis tinuous density gradient) with bindin, the adhesive protein
(376). This phosphatase, named Ci-VSP, is localized in the characteristic of the acrosome (526). Altogether these data
sperm tail plasma membrane. Considering that PIP2 is a suggest that the initial fast, transient [Ca2]i increase trig-
crucial modulator of TRPs and other channels (494) and gered by FSP binding to the sea urchin spermatozoon acti-
Ci-VSP hydrolyzes PIP2 in a voltage-dependent manner vates suPLC- to trigger AR.
(376), possibly SAAF may utilize a TRP channel combined

with Ci-VSP instead of voltage-gated channels. Further Very recently it was shown that binding of FSP to sea urchin
studies are required to confirm this hypothesis. C. intesti- spermatozoa also increases NAADP levels (530) and acti-
nalis is an attractive animal model to study sperm che- vates both sAC and mAC (44). Although both types of ACs
motaxis because it can be genetically manipulated (452). are involved in the sea urchin sperm AR, the contribution of
the sAC is more important (44).
VII. SEA URCHIN ACROSOME REACTION
In L. pictus spermatozoa, binding of EJ or FSP within sec-
onds triggers a transient K-dependent hyperpolarization,
A. Generalities followed by a depolarization. This hyperpolarization pre-
cedes and may lead to the activation of a Ca2-dependent
The sea urchin is a marine organism that has been a pre- Na/H exchange that in turn increases pHi (218). In these
ferred model to study fertilization and AR. Jean C. Dan cells, as in S. purpuratus spermatozoa, [Ca2]i and pHi
originally discovered the AR using sea urchin gametes in increases, and indirectly plasma membrane hyperpolariza-
1952 (131). The AR in sea urchin spermatozoa results in the tion, elevate cAMP levels (45) required for diverse and not
exocytosis of the acrosomal vesicle and the pHi-dependent fully understood signaling events. Tetraethylammonium
polymerization of actin which leads to the formation of the (TEA) or an increase in [K]e (30 40 mM) completely in-
acrosomal tubule (see FIG. 9) (508). This exocytotic process hibit the initial Em changes, and the [Ca2]i, pHi, and cAMP
exposes membrane covered by bindin that will fuse with the increases as well as the AR (215, 454). TEA-sensitive K
egg plasma membrane (43, 134, 383). External Ca2 and its channels (218, 304, 323) have been recorded in planar bi-
uptake are strictly required for sperm AR. After signaling layers with incorporated S. purpuratus sperm plasma mem-
initiation upon contact with the egg vestments, there is a branes. These findings clearly indicate that K channels are
complex and sustained influx of Ca2 required for AR (130, essential for the AR to occur. It is remarkable that little is
226, 456). This reaction is triggered species-specifically by the known about their molecular identity.
EJ, sulfated polysaccharides that surround the oocyte (255,
532). The EJ component that induces AR in sea urchin sper- Planar bilayer reassembly of sea urchin sperm plasma mem-
matozoa is a fucose-sulfated polymer or FSP that interacts brane also revealed the presence of anion channels sensitive
with a sperm plasma membrane receptor (suREJ1). The char- to DIDS. Interestingly, DIDS inhibits the sea urchin sperm
acteristics of the EJ receptor and its homologs are discussed in AR, which suggests that anion channels and transporters
detail below. may participate in this important exocytotic process (371).
In addition, a cAMP-activated cationic channel sensitive to
Binding of EJ or FSP to S. purpuratus sea urchin spermato- Ba2 and two other cationic channels of larger conductance
zoa induces Ca2 and Na influx, and K and H efflux were detected in planar bilayers with incorporated hybrid
(202, 203, 208, 454, 456). These fluxes result in Em changes vesicles shed by the fusion of plasma and acrosomal mem-
(215, 218) and elevations of [Ca2]i (224, 225, 257), pHi branes during AR (460).
(225, 312), [Na]i (437), cAMP (202, 204), and IP3 (153).
These physiological AR agonists also stimulate the activity
of PKA (207, 415), phospholipase D (154), and nitric oxide B. Acrosome Reaction-Associated [Ca2]i
synthase (302). In mammalian spermatozoa, PLC-4 is re- Changes
quired for the ZP- and progesterone-induced AR (186).
PLC- has been cloned (39% identity with bovine PLC2) Sea urchin sperm population experiments with Ca2-
and identified by Western blot analysis (80 kDa) in S. pur- sensitive fluorescent dyes have shown that the FSP-


FIGURE 9. Hypothetical signaling pathway of the AR in sea urchin spermatozoa. The fucose sulfate polymer
from egg jelly (FSP) binds to its receptor (suREJ1) transiently activating, by unknown mechanisms, a TEA-
sensitive K channel (KC). Activation of the K channel hyperpolarizes spermatozoa removing CaV inactivation
and stimulating voltage- and Ca2-dependent Na/H exchange increasing pHi and CaV. A suREJ3/PC2
complex could participate in Ca2 uptake and/or in the depolarization needed to open CaVs. The transient
[Ca2]i elevation may stimulate in parallel PLC- to produce IP3 that would bind to the IP3R, and possibly an
ADP-ribosyl cyclase (ARC) increasing NAADP levels that would activate its receptor (two-pore channel, TPC;
Ref. 572). The S. purpuratus genome contains the predicted sequences for three TPC isofoms (TPC13; Ref.
63). SOCs would then activate (SOC) allowing a sustained [Ca2]i increase fundamental to trigger the AR. As
CatSper channels are present in sea urchin sperm, the pHi increase would activate them. They may contribute
to the [Ca2]i increases associated with the AR in an unknown manner. cAMP elevation (produced by sAC and
mACs) may be needed to stimulate EPAC, to induce fusion that involves SNAREs as in mammalian sperm (344,
85), and is likely to regulate different channels. DIDS-sensitive Cl channels (ClC) possibly regulate osmotic
changes necessary for the AR.
induced AR is accompanied by Ca2 uptake mediated by that their activation is linked. In addition, opening of the
at least two different Ca2 channels. The initial [Ca2]i second channel and the AR are inhibited if the FSP-
increase is fast, transient, and sensitive to the CaV chan- induced pHi increase associated with AR is prevented, for
nel blockers verapamil and DHPs (224, 437) that also instance, by elevating external K to 50 mM. It was
inhibit the AR (454). Approximately five seconds later reported that a lower molecular mass (60 kDa) hydro-
another Ca2 channel (permeable to Mn2 and Na, lyzed form of FSP (hFSP), that increases pHi but cannot
insensitive to DHPs, and pHi dependent) opens, sustain- induce AR, activates the second channel (256).
ing an elevated [Ca2]i for several minutes and allowing
sperm AR (225, 437). It is worth pointing out that inhi- Classical experiments that empty internal stores with thap-
bition of the first channel blocks the second, suggesting sigargin or cyclopiazonic acid hint that the second channel

DARSZON ET AL.
opened during the sea urchin sperm AR could be a SOC 2. CaV channels
(217), as postulated earlier for mammalian spermatozoa
(283, 391, 451). The proposed intracellular Ca2 store was As described in section VIA, two CaV channels were iden-
the acrosome (217, 391). Later experiments showed that tified in S. purpuratus testes. Their sequences were found to
SOC activation resulted in acrosomal exocytosis even in the be most similar to mammalian CaV1.2 and CaV2.3, respec-
absence of actin polymerization (258). The pivotal impor- tively. Antibodies against the CaV2.3 channel and a general
tance of Ca2 efflux from the acrosome to achieve acro- antibody that detects a domain present in all CaV1 and
somal exocytosis for other sperm species has become appar- CaV2 channels labeled the acrosome area of sea urchin sper-
ent in recent years (86, 127, 142, 157, 248, 250, 251, 352, matozoa. It is worth noting that the predicted sequence for
391). the suCaVNL (M_001186699.1) has a polycystic kidney
disease (PKD) domain. Furthermore, the CaV channel an-
In many cell types, IP3, resulting from the hydrolysis of PIP2 tagonists nifedipine (30 M) and nimodipine (30 M),
by PLC, binds to the IP3R, releases Ca2 from intracellular which inhibit the AR, diminished (20 30% of the control)
Ca2 stores such as the endoplasmic reticulum, and acti- the [Ca2]i elevation induced by a K-induced depolariza-
vates SOCs (421). An IP3R-like protein was localized on the tion in valinomycin-treated L. pictus spermatozoa, consis-
sperm acrosome region and less intensely in the flagella tent with the presence of functional CaV channels in sea

(581). As IP3 production had been reported during the AR urchin spermatozoa (223). The functional participation of
(153), it was proposed that IP3 binding to the IP3R in the sea urchin sperm CaV channels in the AR has been suggested
acrosome empties this organelle and activates SOCs. At the by Em and [Ca2]i measurements in sperm populations, and
time it was thought that TRPCs may mediate the sustained by extrapolating the mammalian CaV pharmacology; there-
uptake of Ca2 through the second Ca2 channel opened fore, further work is needed to establish the functional role
during the AR, which could be a SOC (217). In somatic cell of these channels (TABLE 2).
types, DAG stimulates PKC (33), activating a subset of the
TRP family (TRPC3, -6, -7) (54). The direct interaction 3. The inductor of the acrosome reaction
between TRPCs and IP3Rs, as well as depletion of Ca2 and its receptor
from internal Ca2 stores, have been proposed as mecha-
nisms for TRPC activation (reviewed in Ref. 2). As CatSper The natural AR inductor FSP interacts with suREJ1, a
channels are present in sea urchins (433) and they, as well as sperm plasma membrane receptor of 210 kDa with one
the second channel opened by FSP binding to sea urchin transmembrane domain and high homology to the human
sperm, are modulated by pHi (225, 437), it is worth explor- autosomal dominant polycystic kidney disease (ADPKD)
ing if CatSper participates in the AR. protein, PKD1/PC1 (373, 525). Monoclonal antibodies
against suREJ1 induce the AR by opening Ca2 channels
(136, 373, 517).
C. Ca2 Channels Implicated in the Sea
Urchin Sperm Acrosome Reaction Most cases of ADPKD are caused by naturally occurring
mutations in two genetically interacting loci, pkd1 (85%)
1. A Ca2-permeable and voltage-dependent channel and pkd2 (15%) (320). pkd1 encodes a large multispan-
ning membrane protein [PKD1 or polycystin-1 (PC1)],
Initially a voltage-dependent multistate high-conductance while pkd2 encodes a protein [PKD2 or polycystin-2 (PC2)
Ca2 channel was recorded from S. purpuratus flagellar or TRPP2], now known to belong to the TRP superfamily
membranes fused into black lipid membranes (BLMs). The of ion channels (520). The products of the pkd genes play
channel displayed a high main-state conductance of 172 pS key roles in renal and vascular mechanosensory transduc-
in 50 mM CaCl2 solutions with voltage-dependent decay to tion, in primary cilia of renal, nodal, and endothelial cells
smaller conductance states at negative Em. At voltages more (reviewed in Ref. 403).
positive than 40 mV, the main-state open probability had
values of 1.0 and decreased at more negative potentials, Sea urchin spermatozoa possess two additional homologs
following a Boltzmann function with an E0.5 72 mV of suREJ1, suREJ2 and suREJ3. These three proteins con-
and an apparent gating charge value of 3.9. The channel tain the REJ module, a 900 amino acid sequence shared
poorly discriminated for divalent over monovalent cations by PKD1/PC1 and by PKDREJ, a testis-specific protein in
(PCa/PNa 6). La3, Co2, and Cd2 which inhibit the mammals (266) whose function is discussed in the mamma-
EJ-induced AR at millimolar concentrations blocked the lian sperm AR section (497). suREJ2 is present throughout
channel with a similar potency (325). These findings sug- the entire sperm plasma membrane, has two transmem-
gested that this channel could participate in the AR (325). brane segments, is mainly located over the sperm mitochon-
Channels with similar characteristics were transferred di- drion, and seems not to be exposed to the extracellular
rectly from S. purpuratus and L. pictus sea urchin as well as media. Due to its localization, it was proposed that it func-
from mouse intact spermatozoa to BLMs, corroborating tions as an anchor between the mitochondrion and the
the relevance of this Ca2 channel in sperm physiology (42). plasma membrane during spermiogenesis (196). Then

again, suREJ3 is a multidomain orphan receptor with 11 kDa). Since the differential distribution of TRPCs suggests
putative TM segments resembling latrophilins, G protein- that these channels could participate in the AR as well as in
coupled receptors involved in exocytosis (356). Its COOH- sperm motility, functional studies are needed. Unpublished
terminal transmembrane region includes a sequence that is results from our group by C. Wood showed that 20 M
homologous to CaV channels and which has been impli- SKF96365, a TRPC channel blocker, inhibits sea urchin
cated in association with PC2 (424, 568). Interestingly, sperm motility.
both suREJ1 and suREJ3 are found in the plasma mem-
brane over the acrosomal vesicle. Considering the homol- 5. Ca2 stores and Ca2 clearance mechanisms
ogy between the sea urchin sperm suREJ proteins and mam-
malian PKDREJ, it was suggested that both animal groups
Since spermatozoa lack an endoplasmic reticulum, the ac-
may share fertilization signaling pathways (356).
rosome (217, 530), the mitochondrion (8, 105, 230), and
possibly remnants from the nuclear envelope (210) may act
Sea urchin spermatozoa also possess PC2 (suPC2), a six-
as intracellular Ca2 stores. In sea urchin spermatozoa, two
pass transmembrane protein containing a COOH-terminal
Ca2-ATPases have been described, a plasma membrane
cytoplasmic EF hand and coiled-coil domains (382), equiv-
Ca2-ATPase located in the head (229) and a SPCA, sur-
alent to mammalian TRPP2 (PKD2 or PC2) (129) which
prisingly restricted to the mitochondrion. It is known that

interacts with suREJ3 and localizes exclusively as a thin
Ca2-ATPases are responsible for 75% of the Ca2 ex-
band on the sperm plasma membrane overlying the acro-
trusion, and as anticipated, their inhibition completely
somal vesicle (356, 382). There is a long-standing debate
blocks AR, stressing the importance of these enzymes in
about the subcellular localization of TRPP2, as it has been
fertilization. The remaining Ca2 efflux is carried out by a
found in the plasma membrane and in the endoplasmic
Na /Ca2 exchanger (229).
reticulum. In the plasma membrane it can interact for ex-
ample with PC1, TRPC1, TRPC4, and TRPV4. In the en-
It was recently reported that agents that alter the mitochon-
doplasmic reticulum it interacts with IP3Rs, syntaxin, and
drial function via differing mechanisms (CCCP, a proton
RyRs (321, 448, 520). Fascinating questions remain open
gradient uncoupler; antimycin, a respiratory chain inhibi-
as to the function of these proteins in the sea urchin sper-
tor; oligomycin, a mitochondrial ATPase inhibitor; and
matozoa.
CGP37157, a Na/Ca2 exchange inhibitor) induce
[Ca2]i increases that depend on external Ca2. The plasma
It is known that PC1 and PC2 interact to produce Ca2-
membrane permeation pathways activated by the mito-
permeable nonselective currents that transduce extracellu-
chondrial inhibitors are permeable to Mn2, sensitive to
lar stimuli (235). Since channel activities resembling PC1/
SOC blockers (Ni2, SKF96365, and Gd2) (399) as well as
PC2 complexes have been measured in sea urchin sperm
to internal-store ATPase inhibitors (thapsigargin and bis-
membranes (42, 325), REJ and suPC proteins may partici-
phenol) (8). These observations taken together indicate that
pate in the ion fluxes triggered by FSP that lead to the AR. S.
the functional status of the sea urchin sperm mitochondrion
purpuratus sea urchins contain 10 SpREJ proteins within
regulates cell Ca2 homeostasis, including Ca2 influx pos-
the PKD1 gene family with low similarity to each other and
sibly through SOCs.
distinct patterns of expression during embryogenesis but,
except for AR-associated roles of SpREJ1 and SpREJ3, the
A plethora of studies on Ca2 increases in sea urchin sperm
functions of the other SpREJ proteins remain unknown
populations triggering the AR either with the natural in-
(228).
ducer (FSP or EJ) or artificially [i.e., raising external pH to
9, depolarization, nigericin, Ca2 ionophore, etc. (147,
4. Transient receptor potential channels
201)] have been reported. However, it is fundamental to
learn the location and temporal characteristics of the
The S. purpuratus genome contains the predicted sequences
[Ca2]i changes that occur under these conditions, particu-
for TRPC3, TRPC5, and TRPC6. Accordingly, RNAs for
larly on the sperm head, to fully understand the mechanisms
TRPC3 and TRPC6 were found in S. purpuratus testis (G.
that regulate the AR signaling pathways. Therefore, Ca2
Granados-Gonzlez, unpublished data). Using commercial
imaging studies at the single-cell level of sea urchin sperma-
antibodies against the mammalian proteins, our group
tozoa undergoing the AR are urgently needed.
found differential staining in the acrosomal and the mito-
chondrial areas, as well as in the flagella of mature sea
urchin spermatozoa, for TRPC3, TRPC5, and TRPC6. The 6. Cationic channels activated by NAADP
signal for TRPC3 was stronger in the acrosomal area than
in the mitochondrial area and weaker in the flagella. More- NAADP-induced Ca2 release was discovered in the sea
over, consistent with the presence of the mentioned TRPCs urchin egg (93, 311). Indeed, experimental results indicated
in sea urchin spermatozoa, Western blot experiments with that sperm produce NAADP that is injected into the sea
sperm membranes showed different relative mobility bands urchin egg and that it participates in its activation (125,
(TRPC3 109 kDa; TRPC5 115 kDa; TRPC6 140 198). Fertilization experiments in the starfish are consistent

DARSZON ET AL.
with this proposal (366). More recently, it has been found transporters for spermatozoa to achieve their apparently
that NAADP may play a role in the sperm AR, as the acro- simple but crucial goal. Oddly, the only mammalian sperm
some is a lysosome-related organelle and NAADP triggers ionic currents that have been reported by patch clamping of
Ca2 release from lysosome-related organelles of many cell the mouse cytoplasmic droplet (see sects. III and IVD) are
types (126, 198). from the two channels mentioned above, plus those recently
recorded in human spermatozoa that are likely derived
Experiments using Mn2 quenching of fura 2 fluorescence from HV channels (329) (see sect. IVD). However, func-
in sperm vesicles and 45Ca2 uptake in digitonin-permeabi- tional, pharmacological, biochemical, and molecular bio-
lized sea urchin spermatozoa revealed that NAADP directly logical strategies indicate that other channels are present in
regulates acrosomal cation channels (530). Ca2 uptake spermatozoa. It is clear from the research in sea urchin
induced by NAADP was partially inhibited by bafilomycin sperm chemotaxis that more than two Ca2 channels are
(V-ATPase inhibitor) and by glycylphenylalanine 2-naph- required, in addition to the tetraKCNG (see sect. VI), a
thylamide (a lysosomal disruptor), and significantly de- crucial channel for this process that is from a new family
creased by thapsigargin and ionomycin, indicating that the (60, 194). Future experiments will explain why patch-
Ca2 uptake was directly into the acrosome. It is worth clamp recordings in epididymal mouse sperm so far have
pointing out that two-pore channels (TPCs/TPNCs; dis- only revealed a restricted set of ion channels. Are CaVs and

cussed in sect. IIID) may be the acrosomal channels re- other channels functionally silenced during maturation and
sponding to NAADP (530). turned on during capacitation?
We can anticipate fruitful and challenging times ahead for

VIII. CONCLUDING REMARKS research in reproduction and sperm physiology that will
certainly contribute to our better understanding of the won-
These are exciting times for the field of fertilization, as new der of life and hopefully to its preservation in all its trea-
genetic, electrophysiological, and imaging tools are opening sured forms.
new avenues of research and allowing the revision of basic
concepts in gamete physiology. For marine organisms like
the sea urchin, cameras with better temporal and spatial
NOTE ADDED IN PROOF
resolution will facilitate examination of swimming sperma-
During the process of publication, two groups (329a, 484a)
tozoa and their response to chemoattractants in three di-
independently reported that CatSper is the principal Ca2
mensions, and how [Ca2]i fluctuations modulate flagellar
channel activated by progesterone in human spermatozoa.
form. Perhaps soon it will be possible to measure Em
A long-lasting mystery has been solved.
changes in individual spermatozoa, as this would have an
enormous impact on our understanding of many aspects of ACKNOWLEDGMENTS
sperm motility and physiology. Learning more about how
pHi is regulated and its influence on motility, capacitation, We are in debt to Drs. Jorge Carneiro, Chris Wood, Shirley
and the sperm AR is urgently needed. Experiments in mice Ainsworth, Richard Horn, and Victor Vacquier for their
with genetically modified ZP eggs (191) and GFP-labeled suggestions and comments that significantly influenced the
spermatozoa (244) could begin to explore the site and time structure of the review. The reviewers of our work have
when the AR occurs and, with anticipated new probes, made a major contribution that we appreciate enormously.
when and where [Ca2]i increases, and how ZP3 partici- We deeply thank Adan Guerrero for providing FIGURE 8, its
pates and if it has to be glycosylated and where. legend, and valuable comments. We also thank Jose Luis de
la Vega and Yoloxochitl Snchez for technical assistance.
Fertilization is a complex process that generates a unique
individual resulting from the fusion of the male and female Address for reprint requests and other correspondence: A.
gametes. Spermatozoa face a myriad of environmental and Darszon, Departamento de Gentica del Desarrollo y Fisi-
maturational changes as they travel to find the egg. The ologa Molecular, Instituto de Biotecnologa, UNAM,
authors may be wrong, but it seems unlikely that these Avenida Universidad #2001 Col. Chamilpa, CP 62210, Cu-
specialized cells could succeed in their mission with a very ernavaca, Mor., Mxico (e-mail: darszon@ibt.unam.mx).
restricted set of ion channels and transporters. Until today,
from all the ion channels that have been knocked out, only GRANTS
two sperm specific channels, CatSper, a putative Ca2
channel (423), and SLO3, a K channel (416), result in Our work is supported by DGAPA Grants IN211809 (to A.
male infertility, although sperm production is normal. Does Darszon), IN204109 (to C. L. Trevio), IN217409 (to C.
this imply that significant redundancy is essential to safe- Beltran), and IN2211103 (to T. Nishigaki); CONACyT
guard this critical event for life? Alternatively, as has been Grants 49113 (to A. Darszon), 56660 (to T. Nishigaki), and
suggested by some authors (i.e., Ref. 431), nature has 99333 (to C. L. Trevio); and National Institutes of Health
evolved a unique minimal set of specialized channels and Grant R01 HD038082-07A1 (to A. Darszon).

DISCLOSURES 20. Avidan N, Tamary H, Dgany O, Cattan D, Pariente A, Thulliez M, Borot N, Moati
L, Barthelme A, Shalmon L, Krasnov T, Ben-Asher E, Olender T, Khen M, Yaniv I,
Zaizov R, Shalev H, Delaunay J, Fellous M, Lancet D, Beckmann JS. CATSPER2, a
No conflicts of interest, financial or otherwise, are declared human autosomal nonsyndromic male infertility gene. Eur J Hum Genet 11: 497
by the authors. 502, 2003.
21. Baba D, Kashiwabara S, Honda A, Yamagata K, Wu Q, Ikawa M, Okabe M, Baba T.

Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of
cumulus cells and fertilize the egg. J Biol Chem 277: 30310 30314, 2002.
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Calcium and Sperm

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Calcium and Sperm

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Physiol Rev 91: 13051355, 2011

CALCIUM CHANNELS IN THE DEVELOPMENT,

Darszon A, Nishigaki T, Beltran C, Trevio CL. Calcium Channels in the Development,

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Table 1. Expression of Cav channels in sperm cells

Cav1.1 Die at birth 288

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Table 2. Calcium channel blockers inhibit acrosomal reaction (AR)

Calciseptine Cav1 (0.43)# rZP3 (Hu) Inhibition 3 145,# 282

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Mibefradil Cav2.3 (0.4) ZP4 (Hu) Inhibition (1) 90, 120

General calcium channel blocker

Cd2 Cav2.3 (0.8) ZP (Mo) Inhibition (375) 11, 90

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Intracellular calcium channel blocker

2-APB IP3-sensitive (42) SAMMA (Hu) No effect 50 5, 348

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Table 3. Expression of TRP and intracellular channels in sperm cells

IP3R Mo Mo Rt Ha Do Mo (h) Rt (h) Ha (h) Do (h) 515, 539

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2. Protein detection the testis, although morphologically differentiated, mam-

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Ca2 is a fundamental regulatory factor for sperm hyper-

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