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CatSper: The complex main gate of calcium entry in mammalian spermatozoa

Article in Molecular and Cellular Endocrinology · July 2020

DOI: 10.1016/j.mce.2020.110951

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Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Review

CatSper: The complex main gate of calcium entry in

mammalian spermatozoa
Rita Rahban a, b, *, Serge Nef a, b, *
a
Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland
b
Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1206, Geneva, Switzerland

A R T I C L E I N F O A B S T R A C T

Keywords: Calcium ions (Ca2+) are involved in nearly every aspect of cellular life. They are one of the most abundant el­
CatSper ements in mammals and play a vital role in physiological and biochemical processes acting mainly as intracel­
Calcium signaling lular messengers. In spermatozoa, several key functions are regulated by cytoplasmic Ca2+ concentration such as
Mammalian spermatozoa
sperm capacitation, chemotaxis, hyperactive motility, and acrosome reaction. The sperm-specific ion channel
Sperm ion channels
CatSper is the principal calcium channel in sperm mediating the calcium influx into the sperm flagellum and
Fertilization
acting as an essential modulator of downstream mechanisms involved in fertilization. This review aims to pro­
vide insights into the structure, localization, and function of the mammalian CatSper channel, primarily human
and mice. The activation of CatSper by progesterone and prostaglandins, as well as the ligand-independent
regulation of the channel by a change in the membrane voltage and intracellular pH are going to be
addressed. Finally, major questions, challenges, and perspectives are discussed.

1. Introduction
The survival of the animal species relies almost entirely on the ability
of a spermatozoon to fertilize an egg. The male germ cell is a unique
highly specialized cell that is designed to exert its main function outside
of its production site. After being released in the lumen of the seminif­
erous tubules, sperm cells have to accomplish several challenging tasks
before reaching and fertilizing the egg. The journey begins after sper­
matogenesis has been completed culminating in the formation of highly
differentiated spermatozoa that are structurally complete but are still
functionally immature. It is through their transit in the epididymis that
sperms cells complete important maturation steps crucial for fulfilling
their functions. These include sperm chromatin condensation, surface
protein modifications such as the incorporation of cholesterol,
morphological changes, and most importantly sperm motility initiation
and acquisition of coordinated sperm motion (reviewed in Sullivan and
Mieusset (2016)). Membrane remodeling and active glycosylation of
surface components are believed to stabilize the cell in order to remain
in a quiescent state (Cooper, 2007; Sullivan et al., 2019; Sullivan and
Mieusset, 2016; Yeung et al., 1993). These changes are already at this
stage, highly dependent on the exchange of ions and electrolytes
through diverse ion channels (Gupta, 2017). Despite being motile upon
ejaculation, sperms are unable to fertilize the oocyte unless they un­
dergo a “priming” step in the female reproductive tract referred to as
capacitation (Austin, 1952; Chang, 1951). During this process which is
partly dependent on an increase in intracellular calcium concentration
([Ca2+]i), extensive metabolic and physiological changes occur in all
sperm compartments. These changes involve further membrane
remodeling which includes the redistribution, modification, addition, or
removal of proteins acquired during epididymal transit. The female
reproductive tract is indeed rich in albumin that binds to cholesterol and
induces an important cholesterol efflux leading to an increase in the
membrane fluidity. In addition, exposure of sperms to high levels of
bicarbonate present in the female tract induces activation of soluble
adenylate cyclase leading to the production of cyclic adenosine mono­
phosphate (cAMP) and activation of the protein kinase A (PKA) (Gervasi
and Visconti, 2017). The phosphorylation of tyrosine residues by PKA, in
turn, induces membrane hyperpolarization which later activates diverse
calcium channels, key among them are the Cationic channel of Sperm
CatSper and voltage-gated calcium channels Cav leading to an increase
in [Ca2+]i (Darszon et al., 2011; De Jonge, 2017, 2005; Puga Molina
et al., 2018; Visconti et al., 2011). This increase allows the sperm to

* Corresponding authors. Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1206, Geneva,
Switzerland.
E-mail addresses: Rita.Rahban@unige.ch (R. Rahban), serge.nef@unige.ch (S. Nef).

https://doi.org/10.1016/j.mce.2020.110951
Received 12 April 2020; Received in revised form 17 July 2020; Accepted 20 July 2020
Available online 24 July 2020
0303-7207/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: Rita Rahban, Serge Nef, Molecular and Cellular Endocrinology, https://doi.org/10.1016/j.mce.2020.110951
R. Rahban and S. Nef Molecular and Cellular Endocrinology xxx (xxxx) xxx

acquire hyperactive motility, a landmark of sperm capacitation, and an
absolute prerequisite for fertilization. Hyperactivated sperms are char­
acterized by having a high amplitude whip-like asymmetrical tail
bending, are non-progressive in aqueous low-viscosity media, and are
thought to be progressive in the physiological viscosity of the upper
female reproductive tract (de Lamirande et al., 1997; Kirkman-Brown
and Smith, 2011; Mortimer, 1997). Another major consequence of
capacitation is the sperm ability to undergo the acrosome reaction (AR)
as a result of the acquired increase of membrane fluidity. The AR is
characterized by the release of proteolytic enzymes allowing the sperm
to penetrate the Zona Pellucida (ZP) surrounding the oocyte and finally
fertilize it.
Throughout this long journey, the sperm cell must continuously
adapt to new environments to deliver the genetic information needed to
create a new individual. From capacitation and navigation to hyper­
activation and acrosome reaction, calcium ions play a central role by
acting at diverse spatial sites to orchestrate these various events (Pub­
licover et al., 2007, 2008). As the principal calcium channel of sperm,
CatSper is the main gate through which extracellular calcium enters the
sperm flagellum and is therefore vital for sperm fertility (Lishko and
Mannowetz, 2018). Even though the fertilization process is at the center
of creating a new life, it is far from being completely understood.
Improving our understanding of the complexity of the CatSper channel
is not only relevant to better decipher the fertilization process but also to
tackle the increasing male infertility problems on a global level. In
addition, disposing of male contraception alternatives is becoming ur­
gent in a world continuously seeking sexual equity. The scope of this
review is, therefore, to focus on the CatSper channel and its function in
sperm fertilizing capacities.
2. CatSper: the complex, promiscuous and essential calcium
channel of the sperm flagellum
2.1. Structure
Being exclusively expressed in spermatozoa and evolutionarily
conserved among species from mammals to invertebrates, CatSper acts
as a polymodal, chemosensory calcium channel that is unique in con­
trolling a variety of events leading to fertilization. A combination of
mouse genetics, as well as the application of patch-clamp techniques,
revealed that CatSper is the principal Ca2+ channel in mouse sperm
(Kirichok et al., 2006; Lobley et al., 2003; Qi et al., 2007; Quill et al.,
2003; Ren et al., 2001). CatSper is specifically located in the membrane
of the flagellar principal piece and is known to be pH-sensitive and
weakly voltage-dependent (Lishko et al., 2012). In the absence of
extracellular calcium, CatSper also allows the entry of monovalent cat­
ions such as Na+ and Cs+, as well as divalent cations Ba2+ with a much
lower affinity compared to Ca2+ (Sun et al., 2017). The heteromeric
CatSper channel is composed of at least ten subunits with four
pore-forming α subunits (CatSper 1–4) and six auxiliary subunits (β, γ, δ,
ζ, ε and EFCAB9) making it one of the most complex channels ever
described (Fig. 1, A) (Carlson et al., 2003; Chung et al., 2017, 2011;
Darszon et al., 2011; Hwang et al., 2019; Quill et al., 2001; Ren et al.,
2001). The high complexity of CatSper is thought to be required for the
diverse types of stimuli that CatSper can respond to (Visconti et al.,
2011).
2.1.1. Pore-forming α subunits: CatSper 1-4
In 2001, the first member of CatSper (renamed CatSper 1 after the
discovery of other subunits) was identified during sequence homology
searches to the voltage-gated Ca2+ channels Cav (Ren et al., 2001). Very
shortly afterward, CatSper 2 was identified as another pore-forming α
subunit using a signal peptide trapping method (Quill et al., 2001).
CatSper 3 and CatSper 4 were later identified using in silico database
mining (Lobley et al., 2003). Structurally, each of the four distinct
subunits has six transmembrane segments (TM), unlike the Cav that has
24 TM segments in a single polypeptide (Lobley et al., 2003). Impor­
tantly, targeted disruption of each of the four pore-forming α subunits
CatSper 1–4 leads to male infertility caused by loss of channel function
and absence of hyperactivated motility (Carlson et al., 2005, 2003; Qi
et al., 2007; Quill et al., 2003, 2001). It has also been shown that
CatSper2, CatSper3 and CatSper4 co-precipitate with CatSper1 either
from mouse testis lysate or in a heterologous system indicating that
CatSper pore is formed by all four subunits (Qi et al., 2007). Similarly,
humans bearing pathogenic mutations in the CatSper1 and CatSper2
genes suffer from infertility. Like in all other voltage-gated cation
channels, CatSper 1 and CatSper 2 have several positively charged res­
idues in the 4th TM segment. However, CatSper 3 and CatSper 4 have
only two positively charged residues which might explain the mild
voltage dependence of CatSper (Ren et al., 2001). CatSper 1 is different

Fig. 1. Structure of the mammalian CatSper channel and its localization in the sperm flagellum.
A: Located in the principal piece of the sperm flagellum, the heteromeric voltage-dependent, pH-sensitive CatSper channel is composed of ten subunits. The channel
consists of four pore-forming α subunits (CatSper 1–4) as well as five auxiliary subunits: CatSper β (beta), CatSper γ (gamma), CatSper δ (delta), CatSper ζ (zeta) and
CatSper ε (epsilon). A new member of the CatSper complex has been recently identified as EF-hand calcium-binding domain-containing protein 9 or EFCAB9, a
calmodulin-like protein that binds Ca 2+ and acts as a dual calcium and pH sensor for CatSper. B: CatSper channels form functional domains within the principal piece
to enable rapid propagation of the calcium signals along the flagellum. The channel complex is organized in quadrilateral longitudinal nanodomains that form a
unique pattern of four linear threads running down the principal piece of the flagellum.

2
R. Rahban and S. Nef
than the three other α subunits in the fact that it possesses a
histidine-rich large cytoplasmic terminal domain that is speculated to
function as a pH sensor (83 histidines in the 446 amino acid composing
the N terminus) (Ren et al., 2001). The sequence similarity between the
four α subunits is indeed low and ranges between 16 and 22% (Navarro
et al., 2008). Similarly, the interspecies homology of the different
CatSper subunits between mouse and human, is relatively low, ranging
from 50% (CATSPER1) to 69% (CATSPER4) (Brown et al., 2019). This
could explain the significant differences in the function and regulation of
CatSper in these species.
2.1.2. Auxiliary subunits: CatSper β, γ, δ, ζ, ε and EFCAB9
CatSper β was the first reported auxiliary subunit of CatSper that was
identified using database search and qRT-PCR (Liu et al., 2007). It
contains two TM segments with two short cytoplasmic domains and a
large extracellular domain (Fig. 1, A). In contrast, CatSper γ contains
only one TM segment with a large extracellular domain and a short
cytoplasmic tail. The large extracellular domains of CatSper β and
CatSper γ suggest possible channel modulation by external signals such
as ligands and cell-cell interactions during sperm maturation in the
epididymis and/or in the female reproductive tract (Darszon et al.,
2011). CatSper δ has a single TM segment with a large extracellular
domain and a short cytoplasmic tail, very similar to CatSper γ (Chung
et al., 2011). It has been shown that the sperm plasma membrane of
CatSper1 null mice also lacks the other three components of the subunit α
(CatSper 2–4), as well as the auxiliary subunits CatSper β, CatSper γ and
CatSper δ (Babcock, 2007; Chung et al., 2011; Qi et al., 2007). CatSper ζ
and ε co-localize with the rest of the CatSper complex. However, unlike
other CatSper knockout mouse models, lack of CatSperζ disrupts the
quadrilateral longitudinal nanodomain arrangement of CatSper complex
but the channel remains functional and only leads to subfertility in the
mutant mice (Chung et al., 2017, 2011). These studies indicate that
CatSper ζ is involved in the compartmentalization of calcium signaling
and can modulate the calcium mechanism of CatSper. While the quad­
rilateral Ca2+ signaling threads are disrupted in CATSPER 1 knockout
mouse sperm (Chung et al., 2014), they seem not to be in CATSPER 2
deficient human sperm (Schiffer et al., 2020). A new member of the
CatSper complex was recently identified as EF-hand calcium-binding
domain-containing protein 9 or EFCAB9, a calmodulin-like protein that
binds Ca 2+ and acts as a dual calcium and pH sensor (Hwang et al.,
2019). EFCAB9 is an evolutionarily conserved component of the CatSper
channel and is a direct binding partner of CatSperζ. The
EFCAB9-CatSperζ complex is associated with the channel pore and
confers pH-dependent activation and Ca2+ sensitivity thus regulating
the opening and closing of CatSper channel. Null-mice deficient for
CatSperz, Efcab9, or lacking both subunits, were shown to be severely
subfertile with sperms that failed to acquire hyperactivated motility
(Chung et al., 2017; Hwang et al., 2019). The absence of EFCAB9 and/or
CatSperζ renders the channel much less responsive to alkalinization in
the presence of physiologically relevant Ca2+, ultimately making the
channel a less efficient Ca2+ conductor. While CatSper channel orga­
nizes in four doublet bands in the flagellar membrane, loss of Efcab9
alters these doublet tracks as well as sperm surface nanoarchitecture
(Chung et al., 2017, 2014; Hwang et al., 2019). Interestingly, the loss of
CatSperz in mice delayed the targeting of the CatSper complex to the
flagellum, but also led to a periodic thinning and disruption of the linear
signaling domains of CatSper (Chung et al., 2017). This suggests that
CatSperζ could coordinate the insertion of CatSper channel complexes in
the flagellar membrane and the underlying fibrous sheath (FS) proteins
along the axoneme (Fig. 1, B).
2.2. Localization
CatSper channels form functional domains within the principal piece
to enable efficient and fast calcium signal propagation along the fla­
gellum. The principle piece is characterized by the presence of a
3
Molecular and Cellular Endocrinology xxx (xxxx) xxx
microtubule-based axoneme which comprises two central pairs of mi­
crotubules doublets and nine outer microtubule doublets forming the “9
+ 2” pattern. Nine outer dense fibers (ODF) and the FS closely lying
under the plasma membrane surrounding the axoneme as well as two
peripheral longitudinal columns (LC) connected by circumferential ribs
(Fig. 1, B).
Even though it is situated in the principal piece of the sperm fla­
gellum, CatSper mediates calcium influx that propagates within seconds
through the midpiece and the head (Xia et al., 2007). This calcium
propagation is sustained in the sperm head and is thought to be impli­
cated in the acrosome reaction (discussed in paragraph 2.3.3) (Xia and
Ren, 2009a). A mathematical model developed to investigate this
propagation proposes that CatSper mediated calcium influx induces
additional calcium release from internal stores located in the sperm
midpiece (Olson et al., 2010). A similar pattern has been observed when
human sperm are stimulated with odorant such as bourgeonal (Spehr
et al., 2003). The physiological relevance of this calcium propagation, in
addition to being associated with acrosome reaction, is thought to
induce hyperactive motility and regulate chemotaxis (discussed in
paragraph 2.3). Using super-resolution fluorescent microscopy, Chung
and colleagues determined the distribution of CatSper proteins at a
nanometer-scale resolution (Chung et al., 2017, 2014). CatSper channel
complex is organized in quadrilateral longitudinal nanodomains that
form a unique pattern of four linear ‘stripes’ running down the principal
piece of the flagellum (Fig. 1, B). Each of these domains runs along each
side of the two longitudinal columns of the sperm flagella. In addition,
each of these four lines was found to be formed by two rows of CatSper
complexes. It has been hypothesized that within a nanodomain, the
CatSperζ and EFCAB9 subunits connect and interact with the neigh­
boring CatSper complex either in a zigzag manner to the adjacent col­
umn or linearly in the same column to structurally and functionally link
the CatSper complexes (Bystroff, 2018; Vyklicka and Lishko, 2020).
These hypothetical arrangements could be responsible for the synchro­
nization of the CatSper opening as well as a more efficient propagation
of Ca2+ waves over the length of flagella (Fig. 1, B).
2.3. Function
The undeniable importance of CatSper during fertilization lies in the
fact that null mice lacking any of CatSper isoforms are infertile (Jin
et al., 2007; Quill et al., 2003, 2001; Ren et al., 2001) and men with
mutations or deletions in CATSPER1, CATSPER2 and CATSPERE suffer
from infertility (Avenarius et al., 2009; Avidan et al., 2003; Brown et al.,
2018; Hildebrand et al., 2010; Luo et al., 2019; Smith et al., 2013;
Williams et al., 2015; Zhang et al., 2007). To understand what makes
CatSper-deficient males infertile, it is important to summarize our cur­
rent understanding of the role of CatSper in sperm fertilizing capacities.
2.3.1. Rheotaxis, thermotaxis, and chemotaxis
During their journey from ejaculation to fertilization, spermatozoa
must travel a long distance and survive in a variety of environments
characterized by diverse physical and chemical conditions (Fig. 2).
Because of the long distance and the complexity of the convoluted ductal
oviduct, several sperm guidance mechanisms exist employing short- and
long-range cues enabling capacitated sperm to reach the oocyte. Most of
these mechanisms were well described in sea urchin sperm from Arabica
Punctulata but our current knowledge on mammalian guidance mecha­
nisms is still very sparse (Kaupp et al., 2008). Spermatozoa employ at
least three different guidance mechanisms: (i) rheotaxis that refers to the
directed movement against the fluid flow (Miki and Clapham, 2013), (ii)
thermotaxis that refer to directed movement in a temperature gradient
(Bahat et al., 2003) and (iii) chemotaxis that refers to the directed
movement of a cell in a gradient of chemoattractant (Fabro et al., 2002;
Oliveira et al., 1999; Ralt et al., 1994). While chemotaxis is a short-range
mechanism acting at the order of the millimeter, rheotaxis and ther­
motaxis are long-range mechanisms guiding spermatozoa along the
R. Rahban and S. Nef
Fig. 2. The journey of a sperm in the female reproductive tract.
oviduct to the site of fertilization. Interestingly, all three mechanisms of
guidance have been reported to be effective only on capacitated sper­
matozoa, involve hyperactivation and depend, at least in part, on
intracellular Ca2+ suggesting commonality of guidance mechanisms in
mammalian spermatozoa (Boryshpolets et al., 2015; Serafín
Pérez-Cerezales, 2015).
CatSper is ideally positioned to control sperm chemotaxis because
chemotactic movements that guide the spermatozoa to the egg are
dependent on asymmetrical movement (Eisenbach and Giojalas, 2006).
The discovery that both human and rabbit spermatozoa are sensitive to
picomolar concentrations of progesterone secreted by the cumulus cells
surrounding the oocyte lead to the establishment that progesterone
plays the role of a sperm chemoattractant in humans (Fig. 2, B) (Gui­
dobaldi et al., 2008; Teves et al., 2006). This remains however debatable
(Chang and Suarez, 2010) even after the discovery that progesterone act
mainly through CatSper in humans but not in murine sperm (Lishko
et al., 2011; Strünker et al., 2011). Exposure of human sperm to gradi­
ents of progesterone leads to a gradual and transient increase in [Ca2+]i
in the sperm head and calcium oscillations at the base of the flagellum
(Harper et al., 2004). Calcium oscillations often start at low progester­
one levels and their frequency does not change with increasing con­
centration of progesterone. These oscillations regulate flagellar beat but
do not induce the acrosome reaction (Bedu-Addo et al., 2007).
Although progesterone is the best-known chemoattractant,
numerous other compounds secreted either from the cumulus cell sur­
rounding the oocyte or the oocyte itself have been reported to act as
chemoattractants for mammalian spermatozoa. It includes an uniden­
tified hydrophobic non-peptide molecule (Armon et al., 2014), chemo­
kine CCL20 (Caballero-Campo et al., 2014), the natriuretic peptide type
C (NPPC) (Kong et al., 2017) and alluring (Burnett et al., 2011), amongst
other. Whether these compounds are physiologically relevant and
require CatSper and calcium signaling is still unclear.
Concerning rheotaxis and sperm rolling (i.e. the rotation of sperm
around their longitudinal axis), it was initially suggested to require Ca2+
influx via CatSper (Kaupp and Strünker, 2017). Indeed, mouse sperms
lacking CatSper1 fail to undergo rolling and rheotaxis (Miki and Clap­
ham, 2013). However, the hypothesis that rolling and rheotaxis are
mediated by Ca2+ influx is at odds with some data in mice and humans
showing that sperm rolling does not require extracellular Ca2+ (Muschol
et al., 2018) and that exposure of human sperm to flow velocity gradi­
ents does not cause measurable changes in [Ca2+]i (Zhang et al., 2016).
It is only until very recently that a new piece of the puzzle revealed that
rolling and rheotaxis of human and mouse sperm do not require func­
tional CatSper and transmembrane calcium signaling (Schiffer et al.,
2020). These results support the concept that passive biomechanical and
hydrodynamic processes enable sperm rolling and rheotaxis rather than
calcium signaling.
With regards to sperm guidance by thermotaxis, to our knowledge,
4
Molecular and Cellular Endocrinology xxx (xxxx) xxx
there is currently, no data available indicating that CatSper is directly
involved in mammalian thermotaxis. However, the sperm’s sodium
channel TRPV4 which is an important regulator of the activity of
CatSper was found to be activated by warm temperature (37 ◦ C at the
site of fertilization) (Mundt et al., 2018). TRPV4-mediated sodium influx
was shown to induce membrane depolarization, which in turn activates
both Hv1 and CatSper. According to the proposed model, Hv1 then ex­
trudes protons out of the sperm leading to intracellular alkalization and
further activation of CatSper (further discussed in paragraph 2.3 and
Fig. 3). It is, therefore, possible that thermotaxis indirectly regulates
CatSper. Besides, there are indications that the thermosensors for sperm
thermotaxis in mammals may involve opsins (Pérez-Cerezales et al.,
2015).
2.3.2. Hyperactivation
Calcium influx through CatSper and mobilization of intracellular
calcium stores are thought to be one of the key events triggering sperm
hyperactivation (Lishko et al., 2011; Marquez et al., 2007a; Mortimer,
1997; Publicover et al., 2008; Strünker et al., 2011) (Fig. 2, B). After
ovulation, the egg is stored for a short period in the ampulla of the
oviduct, the location where fertilization occurs. The acquisition of hy­
peractive motility is essential for enabling the sperms to detach from the
ciliary oviductal epithelium of the isthmus where they tend to form a
reservoir, arrive at the ampulla at the right time and overcome the
protective vestment of the oocyte (De Jonge, 2005; Demott and Suarez,
1992; Suarez, 2008). The current model explaining this high-amplitude
asymmetrical flagellar bending in mammalian sperm is thought to be
initiated by three main events: the alkalinization of the intracellular
environment, the depolarization of the membrane, and an increase in
the intracellular calcium concentration (Lishko and Mannowetz, 2018).
Comparison of the flagellar beating of CatSper1-null spermatozoa with
wild type revealed that flagellar beating remains symmetric with small
amplitude and low curvature even in capacitating medium (Carlson
et al., 2003). CatSper-deficient sperm cells indeed are unable to acquire
hyperactivation and therefore fail to penetrate the egg’s vestment (Qi
et al., 2007). CatSper-null mouse spermatozoa have also been shown to
be unable to fertilize ZP-intact oocyte but do fertilize ZP-free oocyte,
indicating that CatSper channels are required to allow the sperm to
penetrate the ZP (Ren et al., 2001). The central role of CatSper-mediated
[Ca2+]i increase in inducing hyperactivation was however challenged
and other calcium channels from internal calcium stores by Ca2+
-induced Ca2+ release (CICR) were found to have an important role in
hyperactivation (Alasmari et al., 2013). These calcium stores involve
activation of Inositol 1,4,5-triphosphate receptor (IP3R) and Ryanodine
receptors (RyR) (Alasmari et al., 2013; Darszon et al., 2011; Publicover
et al., 2008). These findings by Alasmari et al. confirm the important
role of CatSper in regulating motility but show that, at least in human
sperm, its role is not directly to support hyperactivation. While
R. Rahban and S. Nef
Fig. 3. Regulation of CatSper channel in human sperm.
mobilization of stored Ca2+ was a potent inducer of hyperactivated
motility, the main functional effect of activating CatSper was to enhance
penetration into viscous medium which was abolished in presence of the
CatSper inhibitor NNC55-0396 (Alasmari et al., 2013). However, in a
more recent finding of a potent and specific CatSper inhibitor (RU1968),
Rennhack et al. found that application of the inhibitor did not suppress
hyperactivation in human sperm suggesting that CatSper is involved in
their ability to acquire the whip-lash movement (Rennhack et al., 2018).
Murine sperms incubated with a calcium-free medium are able to
undergo hyperactivation by mobilization of stored calcium through the
IP3R that were shown to localize in the sperm neck region (Marquez
et al., 2007b). A plasma membrane calcium- ATPase 4 (PMCA 4),
localized in the principal piece of the flagellum, is responsible for
eliminating calcium that is also crucial for sperm motility and acquiring
hyperactivation. Indeed, mitochondrial abnormalities found in PMCA
4-deficient spermatozoa lead to a calcium overload resulting in male
infertility due to defective calcium extrusion (Okunade et al., 2004).
Calcium is therefore not only required for the initiation of hyper­
activation but also to its maintenance by directly regulating components
of the axonemal machinery (Darszon et al., 2007).
2.3.3. Acrosome reaction
The AR allows the sperm, by the release of proteolytic enzymes, to
penetrate the cumulus oophorus and the ZP surrounding the oocyte
(Fig. 2, B) (Jin et al., 2011). This reaction is a prerequisite for sperma­
tozoa to successfully fertilize the egg and it is known to be strictly
dependent on an increase in [Ca 2+]i (Yanagimachi, 1994). Different
theories exist on the nature of the inducers of the human acrosome re­
action physiologically. It is generally believed that AR is triggered by
sperm contact with the zona pellucida, although it has been shown that
most fertilizing spermatozoa in mouse undergo the AR already within
the cumulus oophorus that surrounds the newly ovulated mouse egg
(Bedford, 2011; Inoue et al., 2011; Jin et al., 2011; Sun et al., 2011).
However, it seems that in humans only acrosome intact sperm cells can
bind to the ZP (Liu et al., 2006). While the classical view proposes the
zona pellucida proteins, mainly ZP3, to be the main inductor of
mammalian AR, picomolar concentrations of progesterone secreted by
5
Molecular and Cellular Endocrinology xxx (xxxx) xxx
the cumulus cells is also known to trigger a wave of increase in [Ca 2+]i
and promote priming and AR (Meizel et al., 1997; Uñates et al., 2014).
We can distinguish two types of calcium signaling during this unique,
tightly regulated and irreversible exocytotic process; initial calcium rise
upon binding of sperm to ZP or in response to progesterone activation
followed by another sustained calcium rise due to calcium release from
intracellular stores (Darszon et al., 2011). It has been proposed that
CatSper is responsible for the initial transient Ca2+ rise (Singh and
Rajender, 2015). CatSper mediated calcium influx into the flagellum
then leads to a calcium elevation in the sperm head probably by causing
calcium-dependent release from the intracellular store located in the
sperm neck (Singh and Rajender, 2015; Xia et al., 2007). The role of the
CatSper channel in inducing mammalian AR is still however highly
debatable. In a study showing a tail-to head Ca2+ propagation in mouse
sperm, the CatSper-mediated [Ca2+]i increase seemed not to be required
for AR (Xia et al., 2007). In another more recent study aiming at un­
derstanding the relation between acrosome reaction and
CatSper-mediated calcium influx in human sperm, the effect of proges­
terone on AR was shown to be blunted in the presence of T-type channel
blockers NNC at the same concentrations that inhibit 80% of the
progesterone-stimulated calcium influx (Tamburrino et al., 2014). This
suggests that CatSper-mediated calcium influx could be regulating the
acrosome reaction in humans but more experiments are needed to
establish the role of CatSper in this process (Beltrán et al., 2016).
A: The female reproductive tract is composed of different regions and
the sperm cells must constantly adapt to different environments
throughout their journey from the vagina to the ampulla. The vagina is
the site of deposition of semen and where sperms begin to migrate out of
the seminal plasma and into the cervical mucus. Out of the millions of
spermatozoa deposited in the vagina, only an estimated number of one
hundred capacitated sperms will meet the egg. Capacitation is mainly
induced by exposure of sperms to high concentrations of bicarbonate
(HCO−3 ) and serum albumin. After this first selection process, most of the
sperm cohort continues their way up to the uterus where sperm trans­
port is facilitated via peristaltic contractions and rheotaxis. As they
approach the egg at the ampulla, sperms are exposed to warmer tem­
perature at the site of fertilization (37 ◦ C at the ampulla), a guiding
R. Rahban and S. Nef
mechanism referred to as thermotaxis. B: High concentrations of pro­
gesterone secreted by the cumulus cells forms a gradient that guides the
sperm when in close proximity to the egg through chemotaxis. Proges­
terone, as well as prostaglandins, activate CatSper leading to a rapid
transient increase in intracellular calcium concentration [Ca2+]i. CatS­
per activation is implicated in hyperactivated motility characterized by
a star shaped, non-progressive trajectory in aqueous media. This will
facilitate sperm penetration through the extracellular matrix of cumulus
cells by mechanical shear forces. Sperms will then release their acro­
somal content to digest the Zona Pellucida during the acrosome reaction
and finally fertilize the egg.
2.4. Regulation and cross-talks
The application of patch-clamp techniques to measure CatSper cur­
rents that were adapted to human sperm less than a decade ago paved
the way to the elucidation of the regulation of this highly complex
channel (Kirichok et al., 2006; Kirichok and Lishko, 2011; Lishko et al.,
2011; Strünker et al., 2011). We can divide the mechanisms leading to
CatSper activation in two fashions, the first is ligand-dependent and the
second is ligand-independent. Both mechanisms discussed below are
activated simultaneously and are highly interconnected.
2.4.1. Ligand-dependent pathway
Progesterone: the nanomolar concentrations of progesterone pro­
duced and released by the cumulus cells surrounding the oocyte has long
been known to play a central role in human sperm fertilizing capacities
by acting primarily on acrosome reaction and sperm motility (Black­
more et al., 1990; Forti et al., 1999). However, the unknown mecha­
nisms by which progesterone act on sperm have fascinated and, at the
same time, frustrated reproductive scientists. Progesterone classically
acts in a genomic fashion by binding to a nuclear receptor and initiating
gene transcription, but spermatozoa have a highly condensed genome
and are transcriptionally inactive (Baldi et al., 2009). Various mecha­
nisms have been proposed to explain this “non-genomic” action of
progesterone on sperm. The mechanisms mainly involved G-protein
coupled progestin receptors (mPR), a putative membrane-associated
progesterone steroid receptor PGMRC1 (Progesterone Receptor Mem­
brane Component 1), as well as second messengers such as cAMP, cGMP,
PKA, PKG - induced calcium release from intracellular stores, SOC and
cGMP channels. However, the molecular mechanisms by which pro­
gesterone acts on sperm have remained elusive despite considerable
efforts by the scientific community (Baldi et al., 2009). The discovery of
CatSper as the principal calcium channel in sperm (Ren et al., 2001)
along with the application of patch-clamp techniques in human sperm
have finally led to the discovery of the progesterone’s gateway into
sperm (Publicover and Barratt, 2011). Progesterone, along with an
alkaline pH, stimulates a rapid calcium entry with almost no latency that
is incompatible with a signaling pathway involving metabotropic re­
ceptors and second messengers. Using the patch-clamp technique as well
as optical fluorimetry in sperms loaded with a calcium indicator, pro­
gesterone was shown to induce a biphasic signal of a rapid and transient
calcium increase followed by a slower sustained calcium elevation
(Strünker et al., 2011). The T-type channel blockers, mibefradil, and
NNC, significantly altered the progesterone-induced calcium response.
The constants of half-maximal activation (K1/2) of progesterone-induced
increase in calcium were shifted to lower concentration in capacitated
sperm suggesting that capacitation renders sperm more sensitive to
progesterone (Strünker et al., 2011). Using the patch-clamp, Lishko and
colleagues demonstrated that the addition of progesterone dramatically
increased the amplitude of human monovalent CatSper current (ICatSper)
(Lishko et al., 2011). After the action of progesterone and PGE1 on
human sperm was proved to be “non-genomic” because of the rapid
activation rate, it became clear that progesterone either binds directly to
CatSper or to a receptor that is directly associated with the channel
(Strünker et al., 2011). Progesterone was later proved to act on CatSper
6
Molecular and Cellular Endocrinology xxx (xxxx) xxx
via a membrane endocannabinoid signaling pathway through the α/β
hydrolase domain-containing protein 2 (ABHD2). At rest, CatSper is
thought to be inhibited by the endocannabinoid 2-arachidonoylglycerol
(2-AG) in the flagellar membrane. Upon binding of progesterone,
ABHD2 degrades 2-AG and thereby relieves CatSper from inhibition
(Miller et al., 2016). It is noteworthy to mention that progesterone ac­
tivates CatSper only in human and macaque sperm and does not induce
any increase in [Ca2+]i in mouse sperm (Lishko et al., 2011; Strünker
et al., 2011; Sumigama et al., 2015).
Prostaglandins (PGs): these compounds derived from arachidonic
fatty acids are abundant in the seminal plasma and are secreted by the
oviduct and cumulus cells surrounding the oocyte. In addition to pro­
gesterone, prostaglandin E1 (PGE1) has also been shown to act through
CatSper and increase intracellular calcium concentration in a very
similar biphasic manner with similar amplitude and potentiates the
ICatSper (Lishko et al., 2011; Strünker et al., 2011). The large current
recorded in the presence of PGE1 was fully inhibited by NNC similarly to
the calcium influx measured with fluorimetry. The addition of saturating
concentration of PGE1 (2 μM) to sperm cells already potentiated by
progesterone resulted in a further increase in the amplitude of ICatSper
and vice versa. This suggests that although progesterone and PGE1
activate the same channel, they have two distinct binding sites (Kaupp
and Strünker, 2017; Lishko et al., 2011; Lishko and Mannowetz, 2018;
Miller et al., 2015; Strünker et al., 2011). Consistently, it has been
recently reported that PGs and progesterone act in a highly synergistic
manner (Brenker et al., 2018a). Other PGs also induced an increase in
intracellular calcium concentration but with different signal amplitudes
such as PGE1α and PGE2. The relative effects of ligands activating human
CatSper can be classified as follows: Progesterone > PGE1α ≈ PGE1 >
PGA1 > PGE2 ≫ PGD2 (Lishko et al., 2011; Miller et al., 2015). Similarly
to progesterone, PGE1 does not induce a calcium influx in mouse sperm
underlining once more the important differences between human and
mouse CatSper regulation (Lishko and Mannowetz, 2018). For a long
time, PGs were thought to bind to a prostanoid receptor on human sperm
(Schaefer et al., 1998) but this is incompatible with the biphasic signal of
PG-induced intracellular calcium increase and the pharmacology of the
PGE1 response as measured by fluorimetry (Strünker et al., 2011). The
mechanism of action by which PGs act on CatSper thus remains elusive
and does not involve ABHD2 (Kaupp and Strünker, 2017).
Cyclic nucleotide cAMP and CNG: in intact mouse spermatozoa,
membrane-permeable analogs of cAMP and cGMP (such as 8-Br-cAMP
and 8-Br-cGMP) have been shown to activate CatSper-dependent cal­
cium entry and neither cAMP nor cGMP was shown to elevate [Ca2+]i in
CatSper-null mice (Ren et al., 2001). It was therefore originally proposed
that the CatSper channel can be directly or indirectly activated by cyclic
nucleotides (Ren et al., 2001). The following discoveries could not
demonstrate a direct [Ca2+]i increase in response to cyclic nucleotides,
to photolysis of caged cAMP or in response to bicarbonate-induced
elevation of cAMP (Brenker et al., 2012; Strünker et al., 2011; Wenne­
muth et al., 2003). CatSper-dependent increase in [Ca2+]i induced by
alkalinization and high [K+] was however strongly facilitated by the
presence of bicarbonate that is known to increase cAMP levels through
sAC (Carlson et al., 2003; Xie et al., 2006). In mouse, patch-clamp re­
cordings of CatSper currents were not affected by cyclic nucleotides
(Kirichok et al., 2006) suggesting that the facilitation of CatSper by
cyclic nucleotides is indirect and involves an intermediary signaling
cascade that is disrupted during patch-clamp recordings (Kirichok and
Lishko, 2011). It has been therefore proposed that cAMP facilitates
CatSper-dependent calcium increase via PKA-dependent phosphoryla­
tion (Orta et al., 2018).
Albumin and Zona Pellucida glycoproteins: cholesterol efflux by serum
albumin is a key component in sperm cell capacitation and the influx of
calcium during this process has been linked directly to the presence of
CatSper within sperm cells (Xia and Ren, 2009b). Indeed, Bovine Serum
Albumin (BSA) - induced [Ca2+]i increase was shown by patch-clamp
recordings to be absent in CatSper-deficient sperm. The mechanisms
R. Rahban and S. Nef
by which BSA is coupled to the CatSper channel are still largely un­
known (Ren and Xia, 2010; Sun et al., 2017). Application of solubilized
ZP glycoproteins induces an intracellular calcium increase that is known
to be involved in inducing a change in motility and allowing the release
of the sperm acrosomal content, which is crucial for penetrating the egg.
A suggested role for CatSper in the early phase of ZP-induced increase in
[Ca2+]i was proposed. In CatSper 1- deficient mice sperm, the early
calcium response induced by solubilized ZP glycoproteins is absent and
can be restored with exogenous expression of Green Fluorescent Proteins
(GFP)-tagged CatSper 1 protein (Xia and Ren, 2009a). The mechanisms
involved in transducing ZP-induction to the opening of CatSper channels
are currently unknown (Ren and Xia, 2010).
2.4.2. Ligand-independent pathway
Intracellular alkaline pH: intracellular pH is a critical regulator of
sperm activity, from the moment they are released in the lumen of the
seminiferous tubules until they reach and fertilize the egg. While stored
in the epididymis, the sperms bath in an acidic milieu (pH = 5.5–6.8)
making their intracellular pH (pHi) also acidic (≈6) that helps them
remain quiescent before ejaculation (Acott and Carr, 1984; Jones and
Bavister, 2000). Upon entering the female reproductive tract, sperma­
tozoa are exposed to a higher extracellular pH (pHO) of around 7 due to
the presence of a high concentration of Na+ that elevates the sperm pHi
to about 6.5 as they first become motile (Carr and Acott, 1989; Kirichok
and Lishko, 2011). During their subsequent transit, pHi further increases
as a result of sperm capacitation but remains below pHO. This increase in
pH has been shown to activate the CatSper channel in mice and humans
(Kirichok et al., 2006; Lishko et al., 2011; Strünker et al., 2011).
Application of the patch-clamp technique revealed that intracellular
alkaline pH strongly potentiates the murine CatSper current (ICatSper) by
inducing a seven-fold increase leading to an increase in flagellar [Ca2+]i
and sperm hyperactivation (Kirichok et al., 2006). In humans, intra­
cellular alkalinization by the simple addition of NH4Cl is sufficient to
induce calcium influx and potentiate ICatSper independently of proges­
terone that does not alter pHi (Strünker et al., 2011). Even if murine and
human CatSper1 proteins are highly enriched in histidine which is an
indication of a channel’s pH sensitivity, intracellular alkalinization
alone seems to be sufficient to open murine CatSper but not human
CatSper which also depends on activation by ligands as previously dis­
cussed. One of the major players regulating the intracellular pH in
human but not in mouse sperm is the voltage-gated proton channel Hv1
that is confined to the principal piece of the sperm flagellum and is the
dominant proton conductance channel (Lishko et al., 2010). It allows
only outward transport of protons and is therefore dedicated to inducing
intracellular alkalinization. Human sperm was also shown to hold a
shorter isoform of Hv1 termed Hv1Sper (Berger et al., 2017). It is
generated from Hv1 by the removal of 68 amino acids from the N-ter­
minus by post-translational proteolytic cleavage. In both channels, the
conductance-voltage relationship is determined by the pH difference
across the membrane (ΔpH). However, a constant ΔpH activates only
Hv1Sper but not Hv1. This suggests that cleavage of the N-terminus
regulates pH sensing in Hv1 (Berger et al., 2017). Hv1 is activated by
diverse mechanisms: 1) membrane depolarization, 2) an alkaline
extracellular environment similar to the one in the female reproductive
tract (that can reach up to pH = 8), 3) endocannabinoid anandamide,
and 4) removal of extracellular zinc that is a potent Hv1 blocker (Lishko
et al., 2010; Lishko and Kirichok, 2010). In mouse, the sperm-specific
sodium/proton exchanger (sNHE) is activated by membrane hyperpo­
larization and seems to be responsible for intracellular alkalinization
and consequently CatSper activation (Wang et al., 2007, 2003).
Change in the membrane voltage: the same initial patch-clamp re­
cordings of the CatSper currents also revealed that murine CatSper is
weakly voltage-dependent with a slope factor (k) of 30 compared to a
much steeper k of about 4 in solely voltage-gated ion channels (Kirichok
et al., 2006). The low voltage sensitivity of CatSper can be explained by
the low number of positively charged residues in the S4 transmembrane
7
Molecular and Cellular Endocrinology xxx (xxxx) xxx
domain of the other CatSper subunits in contrast to CatSper 1 (Navarro
et al., 2008; Qi et al., 2007). Human ICatSper recordings revealed
fundamental differences with their rodent’s counterpart. The voltage
dependence of human CatSper is slightly steeper (k = 20) and half
activation voltage in human sperm is strikingly higher with V1/2 human
= +85 mV compared to V1/2 mouse = +11 mV at the same intracellular
pH (7.5) (Kirichok et al., 2006; Lishko et al., 2011). This again suggests
that the regulation of mouse and human CatSper is fundamentally
different. Although the CatSper channel has only weak voltage depen­
dence, it is still very important to the optimal functioning of the channel
since it is directly connected to the channel’s pH sensitivity and the
activation of pH-regulating channels such as Hv1. At low intracellular
pH, the CatSper voltage dependence regulates the channel status by
keeping it closed at sperm physiological membrane potentials (from − 70
mV to 0 mV). An increase in intracellular pH significantly shifts murine
CatSper voltage dependence towards the negative membrane potentials
allowing the channel to open by lower potentials in the physiological
range (Kirichok et al., 2006). The principal regulator of membrane po­
tential is attributed to the potassium permeability that is regulated by
two members of the Slo family of potassium channels: Slo1 and Slo3
(Mannowetz et al., 2013; Navarro et al., 2008; Schreiber et al., 1998).
The principal potassium channel in mouse - Slo3 - is pH-sensitive, cal­
cium-independent, and voltage-sensitive (Brenker et al., 2014; Santi
et al., 2010; Schreiber et al., 1998). Slo3 deficient mice display severely
reduced male fertility (Santi et al., 2010; Zhang et al., 2006). In contrast
to mouse, human sperm potassium current (KSper) is pH-independent,
sensitive to [Ca2+]i and can be inhibited by progesterone (Mannowetz
et al., 2013). In humans, calcium rather than pHi was shown to control
KSper suggesting that the prototypical Ca2+ - regulated K+ channel
regulating human KSper is Slo1. However, human Slo3 is also activated
by calcium rather than alkalinization and is abundantly present in the
human sperm flagellum. This suggests that it is rather Slo3 that repre­
sents the principal K+ channel in human sperm that carries the
Ca2+-activated IKSper current (Brenker et al., 2014). The efflux of po­
tassium through Slo3 was therefore proposed to regulate CatSper by
leading to the membrane hyperpolarization that in turn inhibits the
progesterone-evoked Ca2+ influx through CatSper (Brenker et al., 2014).
2.4.3. CatSper is promiscuous
Under physiological conditions, progesterone, and PGs, together
with membrane voltage and intracellular pH, act collectively to regulate
calcium entry into sperm via the human CatSper. However, CatSper is
also modulated by various endogenous or exogenous compounds and is
known for its promiscuity. In particular, CatSper appears to be regulated
by several endogenous steroids. Pregnenolone sulfate exerts effects
similar to those of progesterone, while physiological concentrations of
testosterone, hydrocortisone and estradiol were first shown to act as
antagonists to CatSper and to reduce or prevent CatSper activation by
progesterone (Mannowetz et al., 2017). However, these results could not
be reproduced by Brenker et al. whose data lead to entirely different
conclusions. Testosterone, hydrocortisone, and estradiol were shown to
enhance CatSper currents, with varying potency and efficacy. In addi­
tion, the three steroids were shown not to antagonize the
progesterone-induced CatSper currents (Brenker et al., 2018b). Simi­
larly, it has been shown that in human follicular fluid unidentified
compounds other than progesterone contribute to the increase of [Ca2+]i
and modulate CatSper activation (Brown et al., 2019). Concerning
exogenous ligands, molecules as diverse as a variety of odorants can
directly act on CatSper without involving G protein-coupled receptors
(GPCRs) or cAMP (Brenker et al., 2012). It has also been shown that
endocrine-disrupting chemicals (EDCs) such as chemical UV filters act
directly on CatSper and induce an increase in [Ca2+]i (Brenker et al.,
2018b; Rehfeld et al., 2017, 2016; Schiffer et al., 2014; Shannon et al.,
2016; Tavares et al., 2013; Yuan et al., 2020). Premature activation of
the CatSper channel by EDCs can potentially desensitize the sperm to
physiological ligands such as progesterone and prostaglandins and may,
R. Rahban and S. Nef
therefore, alter the precisely coordinated sequence of fertilization. With
this in mind, taking advantage of the promiscuous binding to a wide
range of compounds renders CatSper an excellent target for new
non-hormonal contraceptives. Two potential examples were thought to
be good candidates such as the plant triterpenoids Pristimerine and
Lupeol, which are similar in structure to steroid hormones. Both com­
pounds were shown to inhibit CatSper activation by progesterone, sperm
hyperactivation, and slightly reduce basal motility of capacitated human
sperms (Mannowetz et al., 2017; Brenker et al., 2018b). However, these
observations could not be reproduced by Brenker et al. who showed that
both compounds did not affect the progesterone-induced Ca2+ influx
(Brenker et al., 2018b). This indicates that further studies are needed to
address the promiscuous steroid-binding side controlling CatSper.
Regulation of CatSper is critical for proper sperm function and com­
pounds that directly affect CatSper or influence calcium signaling pose a
genuine threat to sperm fertilization potential (Miller et al., 2015).
2.4.4. Integrated model
As sperms enter the female reproductive tract, they are first exposed
to a high concentration of bicarbonate and albumin, two main players in
initiating capacitation by stimulating activation of the sAC/PKA phos­
phorylation pathway and cholesterol efflux, respectively. At the time of
ejaculation, sperms are combined with the seminal fluid, which contains
high enough concentration of zinc to block the activity of the Hv1
channel, by binding to two histidine residues that stabilize the channel
in the closed state (Cherny and DeCoursey, 1999; Lishko and Kirichok,
2010). As sperms ascend from the vagina through the cervix and into the
upper female reproductive tract, divalent zinc ions are chelated by
proteins present in the oviductal fluid resulting in gradual activation of
Hv1 (Lishko et al., 2010; Lu et al., 2008). PKA also phosphorylates Hv1
further activating the channel and potentiating the efflux of protons
(Puga Molina et al., 2018). In order to function properly, CatSper of
human sperm requires three concomitant activation mechanisms: 1)
membrane depolarization, 2) intracellular alkalization and 3) the pres­
ence of progesterone. As sperms approach the site of fertilization, they
are guided by rheotaxis and thermotaxis into warmer temperature at the
ampulla and by chemotaxis through the exposure of a gradient of pro­
gesterone secreted by cumulus cells surrounding the oocyte. The
required conditions for proper CatSper functioning are simultaneously
present; the elevated temperature activates the sperm’s sodium channel
TRPV4 (DSper) inducing a membrane depolarization. This activates Hv1
(as well as CatSper) leading to extrusion of protons by Hv1, intracellular
alkalization, and further activation of CatSper. The presence of sperms
close to a high concentration of progesterone further activates CatSper
promoting the calcium influx and the potassium efflux through KSper.
The latter leads to membrane hyperpolarization and regulation of
CatSper in a negative feedback loop mechanism (Fig. 3).
The complex CatSper channel is composed of ten subunits and is
activated in a ligand dependent fashion – by steroids and prostaglandins-
as well as in a ligand independent fashion via the diversified crosstalk
with other channels and ion exchangers. In order to function properly,
CatSper requires three concomitant activation mechanisms: 1) mem­
brane depolarization, 2) intracellular alkalization and 3) presence of
progesterone. As sperms approach to the site of fertilization, sperms are
exposed to elevated temperature (37 ◦ C) which induce the activation of
sodium channel TRPV4 (also called DSper) leading to membrane de­
polarization following sodium efflux. This activates Hv1 and CatSper
leading to extrusion of protons by Hv1 and further activation of Catsper
due to intracellular alkalization. Concomitantly, progesterone binds to
ABHD2 and releases CatSper inhibition by 2-AG promoting the calcium
influx through CatSper and the potassium efflux through the potassium
channel Slo3 (also called KSper). Influx of calcium induces hyperactive
motility and efflux of potassium, leads to membrane hyperpolarization
and regulation of CatSper in a negative feedback loop mechanism.
8
Molecular and Cellular Endocrinology xxx (xxxx) xxx
3. Major challenges, remaining questions, and perspectives
3.1. Challenge: functional expression of CatSper
The regulation of mammalian CatSper by the various stimuli as well
as the mechanisms by which channel activity is regulated is far from
being completely understood for a variety of reasons. First because the
functional expression of CatSper in any heterologous systems has not yet
been achieved since the channel is resistant to functional expression
(Darszon et al., 2011). This inability to express the channel in other cell
types may be related to the complexity of the multi-subunit constituents
of the channel. Alternatively, it is also possible that this channel requires
a yet unknown essential subunit for the assembly of a functional CatSper
complex. Occurrence and localization of CatSper were indeed shown to
be coordinated with the assembly of the FS proteins along the axoneme
(Chung et al., 2017; Smith et al., 2013). It has been also shown that it is
only after the different CatSper subunits are properly positioned on the
flagellum and are docked to underlying axonemal structures that the
channel becomes functional (Lishko et al., 2012). The last two CatSper
subunits - EFCAB9 and CatSper ζ - were relatively recently identified
(Chung et al., 2017; Hwang et al., 2019). Second, the human and murine
CatSper shows significant differences in both their regulation and ligand
bindings, which certainly reflects an adaptation of sperm to the local
environment of the female reproductive tract. While both murine and
human CatSper are activated at alkaline pH and are weakly
voltage-sensitive (Lishko et al., 2011; Strünker et al., 2011), only the
human and macaque CatSper are sensitive to progesterone (EC50 = 10
nM) and prostaglandins (EC50 = 8 nM) ((Lishko et al., 2012; Sumigama
et al., 2015). Third, the application of the patch-clamp technique was
first made possible in 2006 on mouse sperm and was only introduced to
human sperm less than a decade ago which is relatively a short period of
time (Kirichok et al., 2006; Lishko et al., 2011; Strünker et al., 2011).
Finally, the lack of suitable pharmacological tools has always hindered
the elucidation of CatSper function. It is only until recently that a spe­
cific inhibitor of CatSper (RU1968) was introduced to help better un­
derstand the pharmacological regulation of this complex channel
(Rennhack et al., 2018).
3.2. CatSper as a pharmacological target for male-directed contraception
Due to its unique composition, specific expression in sperm, and
central role in male fertility, the CatSper channel is a prime target for
infertility treatment or male contraception. Early studies have already
explored the contraceptive potential of Anti-CatSper1 IgG (Li et al.,
2009) or the Ca2+ channel blocker HC-056456 that has been shown to
block CatSper channel activity and reversibly prevented hyperactivated
motility of capacitated sperm (Carlson et al., 2009).. We can expect that
in the coming years several studies testing the ability of pharmacological
or natural compounds to act as CatSper agonists or inhibitors will be
identified and made available to the scientific community. Using a
high-throughput screening technique, thousands of ion channels ligands
were assessed for their ability to induce calcium signaling and improve
sperm motility (Martins Da Silva et al., 2017). This allowed the identi­
fication of a phosphodiesterase inhibitor (PDE3), the trequinsin hydro­
chloride, that was shown to act as an efficacious agonist of CatSper, and
increase sperm hyperactivation and penetration into viscous medium
(McBrinn et al., 2019). These pharmacological tools will not only pro­
vide additional insights into the properties, regulation, and role of the
CatSper channel but will also allow us to evaluate their potential as
CatSper-targeting drugs for the treatment of infertility or as
non-hormonal male contraceptives.
3.3. Remaining questions
Although the role of [Ca2+]i signaling in sperm physiology is central,
several questions remain unanswered regarding CatSper and its role in
R. Rahban and S. Nef
mediating sperm function. Future studies should address the precise
organization of the CatSper channels along the four longitudinal nano­
domains in the principal piece of the flagellum and how this arrange­
ment allows synchronization of CatSper opening, making efficient
propagation of Ca2+ waves possible. The translation of these waves into
phosphorylation cascades and structural changes underlying hyper­
activated asymmetrical motility are also still to be clarified. It is also
important to better understand the species differences between human
and rodent CatSper and to identify the other endogenous CatSper li­
gands acting as a chemoattractant for sperm chemotaxis. In terms of
channel regulation, both progesterone and PGs were shown to act on
CatSper synergistically and are equally important in inducing calcium
downstream events leading to fertilization (Brenker et al., 2018a). The
mechanism of action by which progesterone acts on CatSper to elevate
[Ca2+]i has been previously described and is thought to be mediated
through ABHD2 (Miller et al., 2016). However, the mechanism of
CatSper activation by prostaglandins remains elusive and the relation
with the pH-control of the channel is not yet understood. Indeed, the
specific role of both progesterone and PGE1 during fertilization has not
been yet fully established (Baldi et al., 2009). This is mainly due to the
experimental challenges faced in reproducing the chemical composition
of the female reproductive tract on one hand and the lack of appropriate
pharmacological tools to decipher the complex crosstalk of ion channels
involved in regulating sperm physiology on the other hand (Suarez and
Pacey, 2006; Suarez, 2008; Barratt and Publicover, 2012). In addition,
the precise role of membrane potential in regulating CatSper in human
sperm is still not yet elucidated and needs further exploration (Brenker
et al., 2014; Mannowetz et al., 2013). Mechanisms of CatSper silencing
in the male reproductive tract and activation in the female genital tract
also need further investigation.
Acknowledgments
The authors are grateful to the graphic designer Valentin Durand for
assistance with the artwork. This work was supported by the Swiss
Centre for Applied Human Toxicology (SCAHT) and by the Département
de l’Instruction Publique of the State of Geneva.
References
Acott, T.S., Carr, D.W., 1984. Inhibition of bovine spermatozoa by caudal epididymal
fluid: II. Interaction of pH and a quiescence factor 1. Biol. Reprod. 30, 926–935.
https://doi.org/10.1095/biolreprod30.4.926.
Alasmari, W., Costello, S., Correia, J., Oxenham, S.K., Morris, J., Fernandes, L., Ramalho-
Santos, J., Kirkman-Brown, J., Michelangeli, F., Publicover, S., Barratt, C.L.R., 2013.
Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in
human sperm. J. Biol. Chem. 288, 6248–6258. https://doi.org/10.1074/jbc.
M112.439356.
Armon, L., Ben-Ami, I., Ron-El, R., Eisenbach, M., 2014. Human oocyte-derived sperm
chemoattractant is a hydrophobic molecule associated with a carrier protein. Fertil.
Steril. 102, 885–890. https://doi.org/10.1016/j.fertnstert.2014.06.011.
Austin, 1952. The capacitation of the mammalian sperm. Nature 170, 326. https://doi.
org/10.1038/170326a0.
Avenarius, M.R., Hildebrand, M.S., Zhang, Y., Meyer, N.C., Smith, L.L.H., Kahrizi, K.,
Najmabadi, H., Smith, R.J.H., 2009. Human male infertility caused by mutations in
the CATSPER1 channel protein. Am. J. Hum. Genet. 84, 505–510. https://doi.org/
10.1016/J.AJHG.2009.03.004.
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, J.S., 2003. CATSPER2, a human autosomal nonsyndromic male infertility
gene. Eur. J. Hum. Genet. 11, 497–502. https://doi.org/10.1038/sj.ejhg.5200991.
Babcock, D.F., 2007. Wrath of the wraiths of CatSper3 and CatSper4. Proc. Natl. Acad.
Sci. U.S.A. https://doi.org/10.1073/pnas.0610909104.
Bahat, A., Tur-Kaspa, I., Gakamsky, A., Giojalas, L.C., Breitbart, H., Eisenbach, M., 2003.
Thermotaxis of mammalian sperm cells: a potential navigation mechanism in the
female genital tract [1]. Nat. Med. https://doi.org/10.1038/nm0203-149.
Baldi, E., Luconi, M., Muratori, M., Marchiani, S., Tamburrino, L., Forti, G., 2009.
Nongenomic activation of spermatozoa by steroid hormones: facts and fictions. Mol.
Cell. Endocrinol. 308, 39–46. https://doi.org/10.1016/J.MCE.2009.02.006.
Barratt, C.L.R., Publicover, S.J., 2012. Sperm are promiscuous and CatSper is to blame.
EMBO J. 31, 1624–1626. https://doi.org/10.1038/emboj.2012.62.
Bedford, J.M., 2011. Site of the mammalian sperm physiological acrosome reaction.
Proc. Natl. Acad. Sci. U.S.A. https://doi.org/10.1073/pnas.1102296108.
9
Molecular and Cellular Endocrinology xxx (xxxx) xxx
Bedu-Addo, K., Barratt, C.L.R., Kirkman-Brown, J.C., Publicover, S.J., 2007. Patterns of
[Ca2+]i mobilization and cell response in human spermatozoa exposed to
progesterone. Dev. Biol. 302, 324–332. https://doi.org/10.1016/j.
ydbio.2006.09.040.
Beltrán, C., Mata-Martínez, E., Chávez, J.C., Sánchez-Cárdenas, C., 2016. Role of ion
channels in the sperm acrosome reaction. https://doi.org/10.1007/978-3-319-
30567-7_3.
Berger, T.K., Fußhöller, D.M., Goodwin, N., Bönigk, W., Müller, A., Dokani
Khesroshahi, N., Brenker, C., Wachten, D., Krause, E., Kaupp, U.B., Strünker, T.,
2017. Post-translational cleavage of Hv1 in human sperm tunes pH- and voltage-
dependent gating. J. Physiol. 595, 1533–1546. https://doi.org/10.1113/JP273189.
Blackmore, P.F., Beebe, S.J., Danforth, D.R., Alexander, N., 1990. Progesterone and 17α-
hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J. Biol.
Chem. 265, 1376–1380.
Boryshpolets, S., Pérez-Cerezales, S., Eisenbach, M., 2015. Behavioral mechanism of
human sperm in thermotaxis: a role for hyperactivation. Hum. Reprod. 30, 884–892.
https://doi.org/10.1093/humrep/dev002.
Brenker, C., Goodwin, N., Weyand, I., Kashikar, N.D., Naruse, M., Krä Hling, M., Mü
Ller, A., Kaupp, B., Strü Nker, T., 2012. The CatSper channel: a polymodal
chemosensor in human sperm. EMBO J. 31, 1654–1665. https://doi.org/10.1038/
emboj.2012.30.
Brenker, C., Zhou, Y., Müller, A., Echeverry, F.A., Trötschel, C., Poetsch, A., Xia, X.-M.,
Bönigk, W., Lingle, C.J., Kaupp, B., Strünker, T., 2014. The Ca 2+-activated K +
current of human sperm is mediated by Slo3. https://doi.org/10.7554/eLife.01438,
3, 1438.
Brenker, Rehfeld, A., Schiffer, C., Kierzek, M., Kaupp, U.B., Skakkebaek, N.E.,
Strünker, T., Skakkebæk, N.E., Strünker, T., 2018a. Synergistic activation of CatSper
Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting
chemicals. Hum. Reprod. 33, 1915–1923. https://doi.org/10.1093/humrep/dey275.
Brenker, Schiffer, C., Wagner, I.V., Tüttelmann, F., Röpke, A., Rennhack, A., Kaupp, U.B.,
Strünker, T., 2018b. Action of steroids and plant triterpenoids on CatSper Ca2+
channels in human sperm. Proc. Natl. Acad. Sci. U.S.A. https://doi.org/10.1073/
pnas.1717929115.
Brown, S.G., Miller, M.R., Lishko, P.V., Lester, D.H., Publicover, S.J., Barratt, C.L.R.,
Martins, S., Silva, D., 2018. Homozygous in-frame deletion in CATSPERE in a man
producing spermatozoa with loss of CatSper function and compromised fertilizing
capacity. Hum. Reprod. 33, 1812–1816. https://doi.org/10.1093/humrep/dey278.
Brown, S.G., Publicover, S.J., Barratt, C.L.R., Martins da Silva, S.J., 2019. Human sperm
ion channel (dys)function: implications for fertilization. Hum. Reprod. Update 25,
758–776. https://doi.org/10.1093/humupd/dmz032.
Burnett, L.A., Anderson, D.M., Rawls, A., Bieber, A.L., Chandler, D.E., 2011. Mouse
sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich
secretory protein family. Dev. Biol. 360, 318–328. https://doi.org/10.1016/j.
ydbio.2011.09.028.
Bystroff, C., 2018. Intramembranal disulfide cross-linking elucidates the super-
quaternary structure of mammalian CatSpers. Reprod. Biol. 18, 76–82. https://doi.
org/10.1016/j.repbio.2018.01.005.
Caballero-Campo, P., Buffone, M.G., Benencia, F., Conejo-García, J.R., Rinaudo, P.F.,
Gerton, G.L., 2014. A role for the chemokine receptor CCR6 in mammalian sperm
motility and chemotaxis. J. Cell. Physiol. 229, 68–78. https://doi.org/10.1002/
jcp.24418.
Carlson, A.E., Burnett, L.A., del Camino, D., Quill, T.A., Hille, B., Chong, J.A., Moran, M.
M., Babcock, D.F., 2009. Pharmacological targeting of native CatSper channels
reveals a required role in maintenance of sperm hyperactivation. PloS One 4.
https://doi.org/10.1371/journal.pone.0006844.
Carlson, A.E., Quill, T.A., Westenbroek, R.E., Schuh, S.M., Hille, B., Babcock, D.F., 2005.
Identical phenotypes of CatSper1 and CatSper2 null sperm. J. Biol. Chem. 280,
32238–32244. https://doi.org/10.1074/jbc.M501430200.
Carlson, A.E., Westenbroek, R.E., Quill, T., Ren, D., Clapham, D.E., Hille, B., Garbers, D.
L., Babcock, D.F., 2003. CatSper1 Required for Evoked Ca 2 Entry and Control of
Flagellar Function in Sperm.
Carr, D.W., Acott, T.S., 1989. Intracellular pH regulates bovine sperm motility and
protein Phosphorylation1. Biol. Reprod. 41, 907–920. https://doi.org/10.1095/
biolreprod41.5.907.
Chang, 1951. Fertilizing capacity of spermatozoa deposited into the fallopian tubes.
Nature 168, 697–698. https://doi.org/10.1038/168697b0.
Chang, H., Suarez, S.S., 2010. Rethinking the relationship between hyperactivation and
chemotaxis in mammalian Sperm1. Biol. Reprod. 83, 507–513. https://doi.org/
10.1095/biolreprod.109.083113.
Cherny, V.V., DeCoursey, T.E., 1999. pH-Dependent inhibition of voltage-gated H+
currents in rat alveolar epithelial cells by Zn2+ and other divalent cations. J. Gen.
Physiol. 114, 819–838. https://doi.org/10.1085/jgp.114.6.819.
Chung, J.-J., Miki, K., Kim, D., Shim, S.-H., Shi, H.F., Hwang, J.Y., Cai, X., Iseri, Y.,
Zhuang, X., Clapham, D.E., 2017. CatSperζ regulates the structural continuity of
sperm Ca2+ signaling domains and is required for normal fertility. Elife 6. https://
doi.org/10.7554/eLife.23082.
Chung, J.-J., Navarro, B., Krapivinsky, G., Krapivinsky, L., Clapham, D.E., 2011. A novel
gene required for male fertility and functional CATSPER channel formation in
spermatozoa. Nat. Commun. 2, 153. https://doi.org/10.1038/ncomms1153.
Chung, J.J., Shim, S.H., Everley, R.A., Gygi, S.P., Zhuang, X., Clapham, D.E., 2014.
Structurally distinct Ca2+ signaling domains of sperm flagella orchestrate tyrosine
phosphorylation and motility. Cell 157, 808–822. https://doi.org/10.1016/j.
cell.2014.02.056.
Cooper, T.G., 2007. Sperm maturation in the epididymis: a new look at an old problem.
Asian J. Androl. 9, 533–539. https://doi.org/10.1111/j.1745-7262.2007.00285.x.
R. Rahban and S. Nef
Darszon, A., Nishigaki, T., Beltran, C., Treviño, C.L., 2011. Calcium channels in the
development, maturation, and function of spermatozoa. Physiol. Rev. 91,
1305–1355. https://doi.org/10.1152/physrev.00028.2010.
Darszon, A., Treviño, C.L., Wood, C., Galindo, B., Rodríguez-Miranda, E., Acevedo, J.J.,
Hernandez-González, E.O., Beltrán, C., Martínez-López, P., Nishigaki, T., 2007. Ion
channels in sperm motility and capacitation. Soc. Reprod. Fertil. Suppl. 65, 229–244.
De Jonge, C., 2017. Biological basis for human capacitation—revisited. Hum. Reprod.
Update 23, 289–299. https://doi.org/10.1093/humupd/dmw048.
De Jonge, C., 2005. Biological basis for human capacitation. Hum. Reprod. Update 11,
205–214. https://doi.org/10.1093/humupd/dmi010.
de Lamirande, E., Leclerc, P., Gagnon, C., 1997. Capacitation as a regulatory event that
primes spermatozoa for the acrosome reaction and fertilization. Mol. Hum. Reprod.
3, 175–194. https://doi.org/10.1093/molehr/3.3.175.
Demott, R.P., Suarez, S.S., 1992. Hyperactivated sperm progress in the mouse Oviduct1.
Biol. Reprod. 46, 779–785. https://doi.org/10.1095/biolreprod46.5.779.
Eisenbach, M., Giojalas, L.C., 2006. Sperm guidance in mammals — an unpaved road to
the egg. Nat. Rev. Mol. Cell Biol. 7, 276–285. https://doi.org/10.1038/nrm1893.
Fabro, G., Rovasio, R.A., Civalero, S., Frenkel, A., Caplan, S.R., Eisenbach, M., Giojalas, L.
C., 2002. Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed
by a novel directionality-based Assay1. Biol. Reprod. 67, 1565–1571. https://doi.
org/10.1095/biolreprod.102.006395.
Forti, G., Baldi, E., Krausz, C., Luconi, M., Bonaccorsi, L., Maggi, M., Bassi, F.,
Scarselli, G., 1999. Effects of progesterone on human spermatozoa : clinical
implications Les effets de la progestérone sur le spermatozoïde humain : implications
cliniques. Annales d’Endocrinologie.
Gervasi, M.G., Visconti, P.E., 2017. Molecular changes and signaling events occurring in
spermatozoa during epididymal maturation. Andrology. https://doi.org/10.1111/
andr.12320.
Guidobaldi, H.A., Teves, M.E., Uñates, D.R., Anastasía, A., Giojalas, L.C., 2008.
Progesterone from the cumulus cells is the sperm chemoattractant secreted by the
rabbit oocyte cumulus complex. PloS One 3, e3040. https://doi.org/10.1371/
journal.pone.0003040.
Gupta, G., 2017. Sperm maturation in epididymis. In: Male Infertility: Understanding,
Causes and Treatment. Springer Singapore, Singapore, pp. 37–45. https://doi.org/
10.1007/978-981-10-4017-7_4.
Harper, C.V., Barratt, C.L.R., Publicover, S.J., 2004. Stimulation of human spermatozoa
with progesterone gradients to simulate approach to the oocyte. Induction of [Ca2+]
i oscillations and cyclical transitions in flagellar beating. J. Biol. Chem. 279,
46315–46325. https://doi.org/10.1074/jbc.M401194200.
Hildebrand, M.S., Avenarius, M.R., Fellous, M., Zhang, Y., Meyer, N.C., Auer, J.,
Serres, C., Kahrizi, K., Najmabadi, H., Beckmann, J.S., Smith, R.J.H., 2010. Genetic
male infertility and mutation of CATSPER ion channels. Eur. J. Hum. Genet. 18,
1178–1184. https://doi.org/10.1038/ejhg.2010.108.
Hwang, J.Y., Mannowetz, N., Zhang, Y., Everley, R.A., Gygi, S.P., Bewersdorf, J.,
Lishko, P.V., Chung, J.J., 2019. Dual sensing of physiologic pH and calcium by
EFCAB9 regulates sperm motility. Cell 177, 1480–1494. https://doi.org/10.1016/j.
cell.2019.03.047 e19.
Inoue, N., Satouh, Y., Ikawa, M., Okabe, M., Yanagimachi, R., 2011. Acrosome-reacted
mouse spermatozoa recovered from the perivitelline space can fertilize other eggs.
Proc. Natl. Acad. Sci. U.S.A. 108, 20008–20011. https://doi.org/10.1073/
pnas.1116965108.
Jin, J., Jin, N., Zheng, H., Ro, S., Tafolla, D., Sanders, K.M., Yan, W., 2007. Catsper3 and
Catsper4 are essential for sperm hyperactivated motility and male fertility in the
Mouse1. Biol. Reprod. 77, 37–44. https://doi.org/10.1095/biolreprod.107.060186.
Jin, M., Fujiwara, E., Kakiuchi, Y., Okabe, M., Satouh, Y., Baba, S.A., Chiba, K.,
Hirohashi, N., 2011. Most fertilizing mouse spermatozoa begin their acrosome
reaction before contact with the zona pellucida during in vitro fertilization. Proc.
Natl. Acad. Sci. U.S.A. 108, 4892–4896. https://doi.org/10.1073/pnas.1018202108.
Jones, J.M., Bavister, B.D., 2000. Acidification of intracellular pH in bovine spermatozoa
suppresses motility and extends viable life. J. Androl. 21, 616–624. https://doi.org/
10.1002/J.1939-4640.2000.TB02128.X.
Kaupp, U. Benjamin, et al., 2008. Mechanisms of sperm chemotaxis. Annu. Rev. Physiol.
https://doi.org/10.1146/annurev.physiol.70.113006.100654.
Kaupp, U.B., Strünker, T., 2017. Signaling in sperm: more different than similar. Trends
Cell Biol. 27, 101–109. https://doi.org/10.1016/J.TCB.2016.10.002.
Kirichok, Y., Lishko, P.V., 2011. Rediscovering sperm ion channels with the patch-clamp
technique. Mol. Hum. Reprod. https://doi.org/10.1093/molehr/gar044.
Kirichok, Y., Navarro, B., Clapham, D.E., 2006. Whole-cell patch-clamp measurements of
spermatozoa reveal an alkaline-activated Ca2+ channel. Nature 439, 737–740.
https://doi.org/10.1038/nature04417.
Kirkman-Brown, J.C., Smith, D.J., 2011. Sperm motility: is viscosity fundamental to
progress? Mol. Hum. Reprod. 17, 539–544. https://doi.org/10.1093/molehr/
gar043.
Kong, N., Xu, X., Zhang, Y., Wang, Y., Hao, X., Zhao, Y., Qiao, J., Xia, G., Zhang, M.,
2017. Natriuretic peptide type C induces sperm attraction for fertilization in mouse.
Sci. Rep. 7, 39711. https://doi.org/10.1038/srep39711.
Li, H., Ding, X., Guan, H., Xiong, C., 2009. Inhibition of human sperm function and
mouse fertilization in vitro by an antibody against cation channel of sperm 1: the
contraceptive potential of its transmembrane domains and pore region. Fertil. Steril.
92, 1141–1146. https://doi.org/10.1016/j.fertnstert.2008.07.1751.
Lishko, P.V., Botchkina, I.L., Fedorenko, A., Kirichok, Y., 2010. Acid extrusion from
human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell 140,
327–337. https://doi.org/10.1016/J.CELL.2009.12.053.
Lishko, P.V., Botchkina, I.L., Kirichok, Y., 2011. Progesterone activates the principal Ca2
+ channel of human sperm. Nature 471, 387–391. https://doi.org/10.1038/
nature09767.
10
Molecular and Cellular Endocrinology xxx (xxxx) xxx
Lishko, P.V., Kirichok, Y., 2010. The role of Hv1 and CatSper channels in sperm
activation. J. Physiol. 588, 4667–4672. https://doi.org/10.1113/
jphysiol.2010.194142.
Lishko, P.V., Kirichok, Y., Ren, D., Navarro, B., Chung, J.-J., Clapham, D.E., 2012. The
control of male fertility by spermatozoan ion channels. Annu. Rev. Physiol. 74,
453–475. https://doi.org/10.1146/annurev-physiol-020911-153258.
Lishko, P.V., Mannowetz, N., 2018. CatSper: a unique calcium channel of the sperm
flagellum. Curr. Opin. Physiol. 2, 109–113. https://doi.org/10.1016/J.
COPHYS.2018.02.004.
Liu, D.Y., Garrett, C., Baker, H.W.G., 2006. Acrosome-reacted human sperm in
insemination medium do not bind to the zona pellucida of human oocytes. Int. J.
Androl. 29, 475–481. https://doi.org/10.1111/j.1365-2605.2006.00681.x.
Liu, J., Xia, J., Cho, K.-H., Clapham, D.E., Ren, D., 2007. CatSperbeta, a novel
transmembrane protein in the CatSper channel complex. J. Biol. Chem. 282,
18945–18952. https://doi.org/10.1074/jbc.M701083200.
Lobley, A., Pierron, V., Reynolds, L., Allen, L., Michalovich, D., 2003. Identification of
human and mouse CatSper3 and CatSper4 genes: characterisation of a common
interaction domain and evidence for expression in testis. Reprod. Biol. Endocrinol. 1,
53. https://doi.org/10.1186/1477-7827-1-53.
Lu, J., Stewart, A.J., Sadler, P.J., Pinheiro, T.J.T., Blindauer, C.A., 2008. Albumin as a
zinc carrier: properties of its high-affinity zinc-binding site. Biochem. Soc. Trans. 36,
1317–1321. https://doi.org/10.1042/BST0361317.
Luo, T., Chen, H.-Y., Zou, Q.-X., Wang, T., Cheng, Y.-M., Wang, H.-F., Wang, F., Jin, Z.-L.,
Chen, Y., Weng, S.-Q., Zeng, X.-H., 2019. A novel copy number variation in
CATSPER2 causes idiopathic male infertility with normal semen parameters. Hum.
Reprod. 1–10 https://doi.org/10.1093/humrep/dey377.
Mannowetz, N., Miller, M.R., Lishko, P.V., 2017. Regulation of the sperm calcium
channel CatSper by endogenous steroids and plant triterpenoids. Proc. Natl. Acad.
Sci. U.S.A. 114, 5743–5748. https://doi.org/10.1073/pnas.1700367114.
Mannowetz, N., Naidoo, N.M., Choo, S.A.S., Smith, J.F., Lishko, P.V., 2013. Slo1 is the
principal potassium channel of human spermatozoa. Elife. https://doi.org/10.7554/
eLife.01009.001, 2013.
Marquez, B., Ignotz, G., Suarez, S.S., 2007a. Contributions of extracellular and
intracellular Ca2+ to regulation of sperm motility: release of intracellular stores can
hyperactivate CatSper1 and CatSper2 null sperm. Dev. Biol. 303, 214–221. https://
doi.org/10.1016/j.ydbio.2006.11.007.
Marquez, B., Ignotz, G., Suarez, S.S., 2007b. Contributions of extracellular and
intracellular Ca2+ to regulation of sperm motility: release of intracellular stores can
hyperactivate CatSper1 and CatSper2 null sperm. Dev. Biol. 303, 214–221. https://
doi.org/10.1016/j.ydbio.2006.11.007.
Martins Da Silva, S.J., Brown, S.G., Sutton, K., King, L.V., Ruso, H., Gray, D.W., Wyatt, P.
G., Kelly, M.C., Barratt, C.L.R., Hope, A.G., 2017. Drug discovery for male
subfertility using high-throughput screening: a new approach to an unsolved
problem. Hum. Reprod. 32, 974–984. https://doi.org/10.1093/humrep/dex055.
McBrinn, R.C., Fraser, J., Hope, A.G., Gray, D.W., Barratt, C.L.R., Martins da Silva, S.J.,
Brown, S.G., 2019. Novel pharmacological actions of trequinsin hydrochloride
improve human sperm cell motility and function. Br. J. Pharmacol. 176, 4521–4536.
https://doi.org/10.1111/bph.14814.
Meizel, S., Turner, K.O., Nuccitelli, R., 1997. Progesterone triggers a wave of increased
free calcium during the human sperm acrosome reaction. Dev. Biol. 182, 67–75.
https://doi.org/10.1006/dbio.1997.8477.
Miki, K., Clapham, D.E., 2013. Rheotaxis guides mammalian sperm. Curr. Biol. 23,
443–452. https://doi.org/10.1016/J.CUB.2013.02.007.
Miller, M.R., Mannowetz, N., Iavarone, A.T., Safavi, R., Gracheva, E.O., Smith, J.F.,
Hill, R.Z., Bautista, D.M., Kirichok, Y., Lishko, P.V., 2016. Unconventional
endocannabinoid signaling governs sperm activation via the sex hormone
progesterone. Science 352, 555–559. https://doi.org/10.1126/science.aad6887.
Miller, M.R., Mansell, S.A., Meyers, S.A., Lishko, P.V., 2015. Flagellar ion channels of
sperm: similarities and differences between species. Cell Calcium 58, 105–113.
https://doi.org/10.1016/J.CECA.2014.10.009.
Mortimer, S.T., 1997. A critical review of the physiological importance and analysis of
sperm movement in mammals. Hum. Reprod. Update 3, 403–439. https://doi.org/
10.1093/humupd/3.5.403.
Mundt, N., Spehr, M., Lishko, P.V., 2018. TRPV4 is the temperature-sensitive ion channel
of human sperm. Elife 7, 1–20. https://doi.org/10.7554/eLife.35853.
Muschol, M., Wenders, C., Wennemuth, G., 2018. Four-dimensional analysis by high-
speed holographic imaging reveals a chiral memory of sperm flagella. PloS One 13,
e0199678. https://doi.org/10.1371/journal.pone.0199678.
Navarro, B., Kirichok, Y., Chung, J.-J., Clapham, D.E., 2008. Ion channels that control
fertility in mammalian spermatozoa. Int. J. Dev. Biol. 52, 607–613. https://doi.org/
10.1387/ijdb.072554bn.
Okunade, G.W., Miller, M.L., Pyne, G.J., Sutliff, R.L., O’Connor, K.T., Neumann, J.C.,
Andringa, A., Miller, D.A., Prasad, V., Doetschman, T., Paul, R.J., Shull, G.E., 2004.
Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a
major housekeeping function for PMCA1 and a critical role in hyperactivated sperm
motility and male fertility for PMCA4. J. Biol. Chem. 279, 33742–33750. https://
doi.org/10.1074/jbc.M404628200.
Oliveira, R.G., Tomasi, L., Rovasio, R.A., Giojalas, L.C., 1999. Increased velocity and
induction of chemotactic response in mouse spermatozoa by follicular and oviductal
fluids. J. Reprod. Fertil. 115, 23–27. https://doi.org/10.1530/jrf.0.1150023.
Olson, S.D., Suarez, S.S., Fauci, L.J., 2010. A model of CatSper channel mediated calcium
dynamics in mammalian spermatozoa. Bull. Math. Biol. 72, 1925–1946. https://doi.
org/10.1007/s11538-010-9516-5.
Orta, G., de la Vega-Beltran, J.L., Martín-Hidalgo, D., Santi, C.M., Visconti, P.E.,
Darszon, A., 2018. CatSper channels are regulated by protein kinase A. J. Biol. Chem.
293, 16830–16841. https://doi.org/10.1074/jbc.RA117.001566.
R. Rahban and S. Nef
Pérez-Cerezales, S., Boryshpolets, S., Afanzar, O., Brandis, A., Nevo, R., Kiss, V.,
Eisenbach, M., 2015. Involvement of opsins in mammalian sperm thermotaxis. Sci.
Rep. 5 https://doi.org/10.1038/srep16146.
Publicover, S., Barratt, C., 2011. Progesterone’s gateway into sperm. Nature 471,
313–314. https://doi.org/10.1038/471313a.
Publicover, S., Harper, C.V., Barratt, C., 2007. [Ca2+]i signalling in sperm — making the
most of what you’ve got. Nat. Cell Biol. 9, 235–242. https://doi.org/10.1038/
ncb0307-235.
Publicover, S.J., Giojalas, L.C., Teves, M.E., de Oliveira, G.S.M.M., Garcia, A.A.M.,
Barratt, C.L.R., Harper, C.V., 2008. Ca2+ signalling in the control of motility and
guidance in mammalian sperm. Front. Biosci. 13, 5623–5637.
Puga Molina, L.C., Luque, G.M., Balestrini, P.A., Marín-Briggiler, C.I., Romarowski, A.,
Buffone, M.G., 2018. Molecular basis of human sperm capacitation. Front. Cell Dev.
Biol. 6, 72. https://doi.org/10.3389/fcell.2018.00072.
Qi, H., Moran, M.M., Navarro, B., Chong, J.A., Krapivinsky, G., Krapivinsky, L.,
Kirichok, Y., Ramsey, I.S., Quill, T.A., Clapham, D.E., 2007. All Four CatSper Ion
Channel Proteins Are Required for Male Fertility and Sperm Cell Hyperactivated
Motility.
Quill, T.A., Ren, D., Clapham, D.E., Garbers, D.L., 2001. A voltage-gated ion channel
expressed specifically in spermatozoa. Proc. Natl. Acad. Sci. U.S.A. 98,
12527–12531. https://doi.org/10.1073/pnas.221454998.
Quill, T.A., Sugden, S.A., Rossi, K.L., Doolittle, L.K., Hammer, R.E., Garbers, D.L., 2003.
Hyperactivated sperm motility driven by CatSper2 is required for fertilization. Proc.
Natl. Acad. Sci. U.S.A. 100, 14869–14874. https://doi.org/10.1073/
pnas.2136654100.
Ralt, D., Manor, M., Cohen-Dayag, A., Tur-Kaspa, I., Ben-Shlomo, I., Makler, A., Yuli, I.,
Dor, J., Blumberg, S., Mashiach, S., Eisenbach, M., 1994. Chemotaxis and
chemokinesis of human spermatozoa to follicular Factors1. Biol. Reprod. 50,
774–785. https://doi.org/10.1095/biolreprod50.4.774.
Rehfeld, A., Dissing, S., Skakkebæk, N.E., 2016. Chemical UV filters mimic the effect of
progesterone on Ca 2+ signaling in human sperm cells. Endocrinology 157,
4297–4308. https://doi.org/10.1210/en.2016-1473.
Rehfeld, A., Egeberg, D.L., Almstrup, K., Petersen, J.H., Dissing, S., Skakkebæk, N.E.,
2017. EDC IMPACT: chemical UV filters can affect human sperm function in a
progesterone-like manner. Endocr. Connect. 7, 16–25. https://doi.org/10.1530/ec-
17-0156.
Ren, D., Navarro, B., Perez, G., Jackson, A.C., Hsu, S., Shi, Q., Tilly, J.L., Clapham, D.E.,
Perez, G., Jackson, A.C., Hsu, S., Shi, Q., Tilly, J.L., Clapham, D.E., 2001. A sperm
ion channel required for sperm motility and male fertility. https://doi.org/10.1038
/35098027.
Ren, D., Xia, J., 2010. Calcium signaling through CatSper channels in mammalian
fertilization. Physiology 25, 165–175. https://doi.org/10.1152/physiol.00049.2009.
Rennhack, A., Schiffer, C., Brenker, C., Fridman, D., Nitao, E.T., Cheng, Y.-M.,
Tamburrino, L., Balbach, M., Stölting, G., Berger, T.K., Kierzek, M., Alvarez, L.,
Wachten, D., Zeng, X.-H., Baldi, E., Publicover, S.J., Kaupp, U.B., Strünker, T., 2018.
A novel cross-species inhibitor to study the function of CatSper Ca 2+ channels in
sperm. Br. J. Pharmacol. 175, 3144–3161. https://doi.org/10.1111/bph.14355.
Santi, C.M., Martínez-López, P., de la Vega-Beltrán, J.L., Butler, A., Alisio, A.,
Darszon, A., Salkoff, L., 2010. The SLO3 sperm-specific potassium channel plays a
vital role in male fertility. FEBS Lett. 584, 1041–1046. https://doi.org/10.1016/j.
febslet.2010.02.005.
Schaefer, M., Hofmann, T., Schultz, G., Gudermann, T., 1998. A new prostaglandin E
receptor mediates calcium influx and acrosome reaction in human spermatozoa.
Proc. Natl. Acad. Sci. U.S.A. 95, 3008–3013. https://doi.org/10.1073/
pnas.95.6.3008.
Schiffer, C., Müller, A., Egeberg, D.L., Alvarez, L., Brenker, C., Rehfeld, A.,
Frederiksen, H., Wäschle, B., Kaupp, U.B., Balbach, M., Wachten, D., Skakkebaek, N.
E., Almstrup, K., Strünker, T., 2014. Direct action of endocrine disrupting chemicals
on human sperm. EMBO Rep. 15, 758–765. https://doi.org/10.15252/
embr.201438869.
Schiffer, C., Rieger, S., Brenker, C., Young, S., Hamzeh, H., Wachten, D., Tüttelmann, F.,
Röpke, A., Kaupp, U.B., Wang, T., Wagner, A., Krallmann, C., Kliesch, S., Fallnich, C.,
Strünker, T., 2020. Rotational motion and rheotaxis of human sperm do not require
functional CatSper channels and transmembrane Ca 2+ signaling. EMBO J. 1–15.
https://doi.org/10.15252/embj.2019102363.
Schreiber, M., Wei, A., Yuan, A., Gaut, J., Saito, M., Salkoff, L., 1998. Slo3, a novel pH-
sensitive K + channel from mammalian spermatocytes. J. Biol. Chem. 273,
3509–3516. https://doi.org/10.1074/jbc.273.6.3509.
Serafín Pérez-Cerezales, S.B.M.E., 2015. Behavioral mechanisms of mammalian sperm
guidance. Asian J. Androl. 628–632. https://doi.org/10.4103/1008-682X.154308.
Shannon, M., Rehfeld, A., Frizzell, C., Livingstone, C., McGonagle, C., Skakkebaek, N.E.,
Wielogórska, E., Connolly, L., 2016. In vitro bioassay investigations of the endocrine
disrupting potential of steviol glycosides and their metabolite steviol, components of
the natural sweetener Stevia. Mol. Cell. Endocrinol. 427, 65–72. https://doi.org/
10.1016/j.mce.2016.03.005.
Singh, A.P., Rajender, S., 2015. CatSper channel, sperm function and male fertility.
Reprod. Biomed. Online 30, 28–38. https://doi.org/10.1016/J.RBMO.2014.09.014.
Smith, J.F., Syritsyna, O., Fellous, M., Serres, C., Mannowetz, N., Kirichok, Y., Lishko, P.
V., 2013. Disruption of the principal, progesterone-activated sperm Ca2+ channel in
a CatSper2-deficient infertile patient. Proc. Natl. Acad. Sci. U.S.A. 110, 6823–6828.
https://doi.org/10.1073/pnas.1216588110.
Spehr, M., Gisselmann, G., Poplawski, A., Riffell, J.A., Wetzel, C.H., Zimmer, R.K.,
Hatt, H., 2003. Identification of a testicular odorant receptor mediating human
sperm chemotaxis. Science 299, 2054–2058. https://doi.org/10.1126/
science.1080376.
11
Molecular and Cellular Endocrinology xxx (xxxx) xxx
Strünker, T., Goodwin, N., Brenker, C., Kashikar, N.D., Weyand, I., Seifert, R., Kaupp, U.
B., 2011. The CatSper channel mediates progesterone-induced Ca 2+ influx in
human sperm. Nature 471, 382–387. https://doi.org/10.1038/nature09769.
Suarez, S.S., 2008. Regulation of sperm storage and movement in the mammalian
oviduct. Int. J. Dev. Biol. 52, 455–462. https://doi.org/10.1387/ijdb.072527ss.
Suarez, S.S., Pacey, A.A., 2006. Sperm transport in the female reproductive tract. Hum.
Reprod. Update 12, 23–37. https://doi.org/10.1093/humupd/dmi047.
Sullivan, R., Légaré, C., Lamontagne-Proulx, J., Breton, S., Soulet, D., 2019. Revisiting
structure/functions of the human epididymis. Andrology. https://doi.org/10.1111/
andr.12633 andr.12633.
Sullivan, R., Mieusset, R., 2016. The human epididymis: its function in sperm
maturation. Hum. Reprod. Update 22, 574–587. https://doi.org/10.1093/humupd/
dmw015.
Sumigama, S., Mansell, S., Miller, M., Lishko, P.V., Cherr, G.N., Meyers, S.A., Tollner, T.,
2015. Progesterone accelerates the completion of sperm capacitation and activates
CatSper channel in spermatozoa from the rhesus Macaque1. Biol. Reprod. 93 https://
doi.org/10.1095/biolreprod.115.129783.
Sun, T.T., Chung, C.M., Chan, H.C., 2011. Acrosome reaction in the cumulus oophorus
revisited: involvement of a novel sperm-released factor NYD-SP8. Protein Cell.
https://doi.org/10.1007/s13238-011-1022-5.
Sun, X. hong, Zhu, Y. ying, Wang, L., Liu, H. ling, Ling, Y., Li, Z. li, Sun, L. bo, 2017. The
Catsper channel and its roles in male fertility: a systematic review. Reprod. Biol.
Endocrinol. https://doi.org/10.1186/s12958-017-0281-2.
Tamburrino, L., Marchiani, S., Minetti, F., Forti, G., Muratori, M., Baldi, E., 2014. The
CatSper calcium channel in human sperm: relation with motility and involvement in
progesterone-induced acrosome reaction. Hum. Reprod. 29, 418–428. https://doi.
org/10.1093/humrep/det454.
Tavares, R.S., Mansell, S., Barratt, C.L.R., Wilson, S.M., Publicover, S.J., Ramalho-
Santos, J., 2013. p,p’-DDE activates CatSper and compromises human sperm
function at environmentally relevant concentrations. Hum. Reprod. 28, 3167–3177.
https://doi.org/10.1093/humrep/det372.
Teves, M.E., Barbano, F., Guidobaldi, H.A., Sanchez, R., Miska, W., Giojalas, L.C., 2006.
Progesterone at the picomolar range is a chemoattractant for mammalian
spermatozoa. Fertil. Steril. 86, 745–749. https://doi.org/10.1016/j.
fertnstert.2006.02.080.
Uñates, D.R., Guidobaldi, H.A., Gatica, L.V., Cubilla, M.A., Teves, M.E., Moreno, A.,
Giojalas, L.C., 2014. Versatile action of picomolar gradients of progesterone on
different sperm subpopulations. PloS One 9, e91181. https://doi.org/10.1371/
journal.pone.0091181.
Visconti, P.E., Krapf, D., de la Vega-Beltrán, J.L., Acevedo, J.J., Darszon, A., 2011. Ion
channels, phosphorylation and mammalian sperm capacitation. Asian J. Androl. 13,
395–405. https://doi.org/10.1038/aja.2010.69.
Vyklicka, L., Lishko, P.V., 2020. Dissecting the signaling pathways involved in the
function of sperm flagellum. Curr. Opin. Cell Biol. https://doi.org/10.1016/j.
ceb.2020.01.015.
Wang, D., Hu, J., Alexandru Bobulescu, I., Quill, T.A., McLeroy, P., Moe, O.W.,
Garbers, D.L., 2007. A Sperm-specific Na/H Exchanger (sNHE) Is Critical for
Expression and in Vivo Bicarbonate Regulation of the Soluble Adenylyl Cyclase
(sAC).
Wang, D., King, S.M., Quill, T.A., Doolittle, L.K., Garbers, D.L., 2003. A new sperm-
specific Na+/H+ exchanger required for sperm motility and fertility. Nat. Cell Biol.
5, 1117–1122. https://doi.org/10.1038/ncb1072.
Wennemuth, G., Babcock, D.F., Hille, B., 2003. Calcium clearance mechanisms of mouse
sperm. J. Gen. Physiol. 122, 115–128. https://doi.org/10.1085/jgp.200308839.
Williams, H.L., Mansell, S., Alasmari, W., Brown, S.G., Wilson, S.M., Sutton, K.A.,
Miller, M.R., Lishko, P.V., Barratt, C.L.R., Publicover, S.J., Martins Da Silva, S., 2015.
Specific loss of CatSper function is sufficient to compromise fertilizing capacity of
human spermatozoa. Hum. Reprod. 30, 2737–2746. https://doi.org/10.1093/
humrep/dev243.
Xia, J., Reigada, D., Mitchell, C.H., Ren, D., 2007. CATSPER channel-mediated Ca2+
entry into mouse sperm triggers a tail-to-head Propagation1. Biol. Reprod. 77,
551–559. https://doi.org/10.1095/biolreprod.107.061358.
Xia, J., Ren, D., 2009a. Egg coat proteins activate calcium entry into mouse sperm via
CATSPER Channels1. Biol. Reprod. 80, 1092–1098. https://doi.org/10.1095/
biolreprod.108.074039.
Xia, J., Ren, D., 2009b. The BSA-induced Ca(2+) influx during sperm capacitation is
CATSPER channel-dependent. Reprod. Biol. Endocrinol. 7, 119. https://doi.org/
10.1186/1477-7827-7-119.
Xie, F., Garcia, M.A., Carlson, A.E., Schuh, S.M., Babcock, D.F., Jaiswal, B.S., Gossen, J.
A., Esposito, G., van Duin, M., Conti, M., 2006. Soluble adenylyl cyclase (sAC) is
indispensable for sperm function and fertilization. Dev. Biol. 296, 353–362. https://
doi.org/10.1016/J.YDBIO.2006.05.038.
Yanagimachi, 1994. Mammalian fertilization. In: The Physiology of Reproduction.
Yeung, C.H., Cooper, T.G., Oberpenning, F., Schulze, H., Nieschlag, E., 1993. Changes in
movement characteristics of human spermatozoa along the length of the
Epididymis1. Biol. Reprod. 49, 274–280. https://doi.org/10.1095/
biolreprod49.2.274.
Yuan, Y., Ding, X., Cheng, Y., Kang, H., Luo, T., Zhang, X., Kuang, H., Chen, Y., Zeng, X.,
Zhang, D., 2020. PFOA evokes extracellular Ca2+ influx and compromises
progesterone-induced response in human sperm. Chemosphere 241. https://doi.org/
10.1016/j.chemosphere.2019.125074.
R. Rahban and S. Nef
Zhang, X., Zeng, X., Lingle, C.J., 2006. Slo3 K + channels: voltage and pH dependence of
macroscopic currents. J. Gen. Physiol. 128, 317–336. https://doi.org/10.1085/
jgp.200609552.
Zhang, Y., Malekpour, M., Al-Madani, N., Kahrizi, K., Zanganeh, M., Lohr, N.J.,
Mohseni, M., Mojahedi, F., Daneshi, A., Najmabadi, H., Smith, R.J.H., 2007.
12
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Molecular and Cellular Endocrinology xxx (xxxx) xxx
Sensorineural deafness and male infertility: a contiguous gene deletion syndrome.
J. Med. Genet. 44, 233–240. https://doi.org/10.1136/jmg.2006.045765.
Zhang, Z., Liu, J., Meriano, J., Ru, C., Xie, S., Luo, J., Sun, Y., 2016. Human sperm
rheotaxis: a passive physical process. Sci. Rep. 6, 23553. https://doi.org/10.1038/
srep23553.