ion channels in sperm physiology and male fertility and infertility
TRANSCRIPT
1
Ion channels in sperm physiology, male fertility and infertility
Kamla Kant Shukla1, 3, Abbas Ali Mahdi2, Singh Rajender1
1Division of Endocrinology, Central Drug Research Institute (CSIR), Lucknow, UP, India,
2Department of Biochemistry, C.S.M. Medical University, Lucknow UP, India
3Reproductive Physiology & Control, Department of Animal Science and Technology, Chung Aug University, Ansung Gyeonggi-Do 456-756, Korea
Address for correspondence / reprint requests:
Dr. Singh Rajender
Scientist
Division of Endocrinology
Central Drug Research Institute
Lucknow, U.P., India - 226001
E-mail: [email protected]
Phone No. +91-522-2613894-Extn. 4395
Fax No. +91-522-2623405
Running title: Ion channels in male fertility and infertility
Published-Ahead-of-Print on March 22, 2012 by Journal of Andrology
Copyright 2012 by The American Society of Andrology
1
Abstract 1
Ion channels regulate the membrane potential and intracellular ionic concentration and thus 2
serve a central role in various cellular processes. Several ion channels have been identified in 3
the germ cells including sperm, emphasizing their importance in male fertility and 4
reproduction. The molecular mechanism of ion transport and the nature of ion channels 5
involved have begun to emerge only recently despite the fact that several ligand and voltage 6
gated channels had been identified and localized on sperm. The presence of catsper gene 7
family channels (CatSpers 1-4), proton voltage gated ion channel (Hv1), potassium voltage 8
gated ion channel (SLO3/KCNU1), sodium voltage gated channel (Nav 1.1-1.9) and the 9
members of transient receptor potential (TRP) channel family suggest indispensable role of 10
ion channels in sperm physiology and fertility potential. Ion channels are the key players in 11
very important processes such as capacitation and acrosome reaction which are critical steps 12
in sperm physiology preparing it for fertilization. For example, CatSpers, Hv1, Slo3, TRPC 13
family members have been proposed to participate in acrosome reaction, thereby making 14
them most important for sperm fertility. Similarly, Nav channels could play a crucial role in 15
the non-capacitated sperm and in the initial capacitation steps. The role of ion channels seems 16
indispensable for sperm fertility as evident from studies on animal models; however, the 17
functional defects in infertile human males await further exploration. The present article 18
brings an update on the role of ion channels in sperm physiology, male fertility and infertility. 19
20
Key words: Ion channels, sperm physiology, sperm capacitation, catsper gene 21
22
23
2
Introduction 1
In mammals, internal fertilization is practiced by depositing the sperm at different sites in the 2
female reproductive tract. Upon deposition in the female tract, the waiting period for the 3
sperm in different mammalian species lasts from few minutes to few days or even months 4
(Suarez and Pacey, 2006). The site of sperm deposition and the time of sperm activation vary 5
widely across species (Suarez and Pacey, 2006). Within minutes of vaginal deposition, 6
human sperm begin to leave the seminal fluid and swim into the cervical canal (Sobrero and 7
MacLeod, 1962). The initial journey of sperm towards the fertilization site is supported by 8
the ovarian contractions of the myometrium (Suarez and Pacey, 2006). In humans, contractile 9
activity of uterine muscle may draw sperm and watery mid-cycle mucus from the cervix into 10
the uterus. The crucial process of fertilization needs to be far from being random and special 11
intricate mechanisms ensuring directed movement of only healthy and potent spermatozoa 12
must exist. For example, once inside the uterus, cervical mucus has been proposed to present 13
a barrier to the sperm such that only actively swimming sperm with proper hydrodynamic 14
profile are able to penetrate the thick mucus. This serves as a means of sperm selection 15
(Hanson and Overstreet, 1981; Katz et al, 1997), ensuring quality control. 16
17
Strong evidence backs the view that the journey of sperm towards oocyte is a guided event 18
and an orchestrated series of changes prepare it for fertilization. There is evidence of 19
existence of at least two systems operating within the fallopian tubes. The sperm are guided 20
in long-range by thermotaxis and it has been shown that capaciated rabbit sperm tend to swim 21
towards warmer temperatures (Bahat et al., 2003). Experimental evidence suggests that tubal 22
ampulla is warmer by 2 °C in comparison to tubal isthmus (Bahat et al., 2003). At some point 23
in the female tract, most likely in the fallopian tubes, sperm become hyperactivated. The 24
3
pattern of asymmetrical motion with lower frequency and higher amplitude, immediately 1
after spermatozoa are exposed to the environment of the female reproductive tract is termed 2
as hyper-activation (Ho and Suarez, 2001; Suarez and Ho, 2003). Hyper-activation helps 3
sperm swim through viscoelastic substances such as mucus in the tubal lumen and the 4
extracellular matrix of the cumulus oophorus. Once in the tubal ampulla, at a closer proximity 5
to the oocyte, chemotectic mechanisms may guide sperm closer to the oocyte (Eisenbalch, 6
1999; Babcock, 2003). At the end of its journey, upon contact with zona-pellucida, acrosome 7
reaction takes place, followed by fertilization. 8
9
Given a strong role of female reproductive tract in preparing sperm for fertilization, the 10
sperm cannot be oblivious to the external environment, thus ensuring that it fuses with no 11
other cell but the egg. For directed movement, the sperm must sense changes in the ionic or 12
osmotic environment and thus ion channels have an important role to play (Morisawa, 1994; 13
Darszon et al, 1999). Today we know two very important processes during this phase of 14
journey. The sperm first undergo capacitation which involves biochemical, bio-physical and 15
metabolic modifications of all sperm domains. Capacitation, though can be easily achieved in 16
the laboratory, even in simple fluid milieu (Harrison and Gadella, 2005), overwhelming 17
evidence has been provided that in vivo sperm capacitation is actively and progressively 18
coordinated within succeeding regions of the female genital tract and could be influenced by 19
ovulation (Rodriguez-Martinez, 2007). The importance of ion channels in sperm physiology 20
and function is also highlighted by the presence of numerous ion channels on these highly 21
differentiated and specialized cells aimed only at delivering haploid DNA to the oocyte. The 22
second process, acrosome reaction, takes place at the end of the journey upon contact with 23
the occyte. 24
4
1
The etiology of infertility remains uncertain in more than 50% of the couples despite all 2
normal semen parameters. These cases are labelled idiopathic, hampering both diagnosis and 3
treatment. Indispensable role of ion channels in sperm physiology and fertility propels us to 4
propose that loss of ion channels function could cause abnormal capacitation, loss of 5
hyperactivation and acrosome reaction, resulting in failure of fertilization despite all other 6
semen parameters being normal. Therefore, study of ion channels in sperm promises to open 7
a new horizon for identification of factors determining sperm fertility and causes of 8
infertility. Understanding of the molecular mechanisms of the ion channels regulating sperm 9
motility would pave the way to identify novel targets for treating infertility and may also help 10
in designing functional novel male contraceptives. In the present article, we have reviewed 11
the literature on ion channels in mammalian sperm, their role in male fertility and correlation 12
with infertility with particular reference to humans. 13
14
Classification of ion channels 15
Ion channels may be classified by gating, i.e. what opens and closes the channel. Voltage-16
gated ion channels open or close depending on the voltage gradient across the plasma 17
membrane. For most voltage-gated ion channels, the pore-forming subunits are called the α 18
subunit, while the auxiliary subunits are denoted as β, γ and so on (Catterall et al, 1999). 19
Some channels permit the passage of ions based solely on their charge of positive (cation) or 20
negative (anion). However, the archetypal channel pore is just one or two atoms wide at its 21
narrowest point and is selective for specific species of ions, such as sodium or potassium. 22
These ions move through the channel pore single file nearly as quickly as the ions move 23
through free fluid. Voltage-gated ion channels are generally classified based on the selective 24
5
conductivity of ions, such as Na+, Ca2+ and K+ channels. Ligand gated channels open or close 1
in response to binding of a ligand to the channel. Ligand-gated ion channels are classified on 2
the basis of the primary signalling transmitter, such as acetylcholine, 5-hydroxy tryptamine 3
(5-HT) and γ-amino butyric acid (GABA) (Collingridge et al, 2009). A detailed account of 4
the ion channels and their role in fertility is given in the following sections. 5
6
CatSper ion channel family (Catsper 1-4) 7
In mammalian sperm, expression of putative 6 trans-membrane ion channel-like proteins, 8
termed CatSper (cation channel of sperm) having four members CatSper1, CatSper2, 9
CatSper3, and CatSper4 has been reported (Quill et al, 2001; Ren et al, 2001; Lobley et al, 10
2003). CATSPER 1 is located on human chromosome 11q12.1, CATSPER 2 on 15q15.3, 11
CATSPER 3 on 5q31.1, and CATSPER 4 on 1p35.3. The first member of this family, 12
CatSper1, was identified during sequence homology search to the voltage-gated Ca2+-13
selective channels (CaV1-3) (Ren et al, 2001), followed by identification of other three 14
members of this family (Navarro et al, 2008). The four CatSper proteins have high homology 15
but relatively low sequence identity in the transmembrane regions, ranging from 16 to 22% 16
(Navarro et al, 2008). The expression pattern of CatSper genes suggests their crucial role in 17
sperm physiology and fertility. Northern blot analysis reported that CatSper 1,2,3,4 mRNA 18
was present exclusively in mouse and human testis (Quill et al, 2001; Ren et al, 2001; Jin et 19
al, 2005; Qi et al, 2007). Temporal expression has been explored using in situ hybridization 20
reporting that CatSper 2 transcription begins early in the spermatogenesis (pachytene 21
spermatocytes) (Quill et al, 2001; Schultz et al, 2003) while CatSper 1 (Ren et al, 2001), 3 22
and 4 (Schultz et al, 2003; Jin et al, 2005; Qi et al, 2007) are transcribed only during late 23
stages (spermatids). More sensitive technique, real time PCR, has also confirmed this pattern 24
6
of expression, reporting CatSper2 expression at post-natal day 8, CatSper 3 and 4 at post-1
natal day 15 and CatSper1 at post-natal day 18 (Li et al, 2007). 2
3
In general, a complete ion channel is composed of pore-forming proteins and one or more 4
auxiliary sub-units. Unlike the composition of NaVs and CaVs, the subunits of CATSPER 5
channels are not well studied. As discussed below, all four CATSPER units are required for 6
functional alkalization-activated current; therefore, the pore is thought to be a tetramer of four 7
CATSPERs (Qi et al., 2007). In addition, the channel complex contains the multiple TM-8
spanning protein CATSPERB (previously called CATSPERβ) (Liu et al., 2007). Wang et al., 9
identified a novel single-TM protein CATSPERG associated with the CATSPER complex. 10
Thus, the CATSPER protein complex consists at least three TM proteins: the pore-forming 11
six-TM CATSPER1–4, a predicted two-TM CATSPERB (Liu et al., 2007), and the single-12
TM CATSPERG (Wang et al., 2009). This structure is comparable to that of other ion 13
channels such as CaVs, NaVs, and KVs. Protein purification experiments did not detect other 14
major proteins in the CATSPER1-containing complex (Liu et al., 2007). The authors 15
suggested that some other proteins might have a role in the complex, but they may have a 16
weak association with CATSPER1 that does not survive the purification with detergents (Qi 17
et al., 2007). 18
19
To figure out the effect of the loss of CatSper, deletion mice models have been developed for 20
all the four CatSper genes. Past work with CatSper1 null sperm indicates that a critical lesion 21
in hyperactivation underlies the infertility phenotype and is associated with an absence of 22
depolarization-evoked Ca2+ entry (Carlson et al., 2005). In one of the initial reports, Ren et al, 23
(2001) demonstrated that CatSper is required for normal sperm motility and egg penetration. 24
The authors generated CatSper-/- mice and a closer examination of live sperm under light 25
7
microscopy revealed a significant difference between the mutant and wild type sperm. Sperm 1
from wild-type mice displayed vigorous beating in the tail region and progressive directed 2
movement. By contrast, CatSper-/- sperm were sluggish and displayed less directed 3
movements. Most notably, mutant sperm lacked the vigorous beating and bending in the tail 4
region. Computer-assisted sperm analysis showed that the mutant sperm's main motility 5
parameters of path velocity, progressive velocity and track speed were impaired significantly. 6
The authors concluded that disrupting the CatSper gene results in markedly reduced sperm 7
motility. The authors also performed in vitro fertilization (IVF) assays to test CatSper-/- 8
sperm's ability to fertilize eggs. CatSper-/- sperm were unable to fertilize any egg, whereas 9
wild-type sperm fertilized 81% of eggs. Some CatSper-/- sperm adhered to the eggs but did 10
not seem to be able to penetrate the eggs, possibly owing to impaired motility. During the 11
course of natural fertilization, sperm penetrate the surrounding layer of zona pellucida 12
proteins and fuse to the egg's plasma membrane. To examine whether the mutant sperm 13
retained the ability to fuse with the plasma membrane and activate fertilization, the authors 14
incubated wild-type and mutant sperm with eggs whose outer layers had been enzymatically 15
removed (zona-pellucida-free eggs). Under such conditions, sperm from both CatSper+/+ and 16
CatSper-/- mice were capable of fertilizing the eggs. It was concluded that CatSper is 17
required for the sperm to be able to penetrate the egg’s outer layers, but not for egg activation 18
(Ren et al, 2001). 19
20
21
It was demonstrated using gene knockout mice that CatSper1 and CatSper2 are essential for 22
the hyperactivation (Ren et al, 2001; Carlson et al, 2003; Quill et al, 2003; Zhang et al, 2006; 23
Marquez et al, 2007). Male mice deficient in either the Catsper1 or the Catsper2 gene are 24
completely infertile, and neither Catsper1-null nor Catsper2-null sperm display 25
8
hyperactivation in capacitating conditions. Similarly, adult Catsper3_/_ and Catsper4_/_ 1
males bred with adult wild type females for more than 4 months did not sire any offspring, 2
despite their normal mating behavior, whereas their heterozygous littermates produced pups 3
with normal litter size and intervals (Jin et al, 2007). Therefore, both Catsper3_/_ and 4
Catsper4_/_ mice are completely infertile, despite their normal sperm counts and initial 5
motility. In the capacitating medium, wild type spermatozoa started to develop 6
hyperactivation after 30 minutes of incubation at room temperature, whereas Catsper 3-null 7
or Catsper 4-null spermatozoa still displayed initial motility during 2 hours of incubation. 8
This result implies that functional CatSper3 and CatSper4 proteins are required for sperm 9
hyperactivation during capacitation. Interestingly, when transferred to Ca2+ free medium, 10
wild type sperm became motionless within 15 min, whereas Catsper3-null or Catsper4-null 11
spermatozoa still displayed a weaker initial motility, suggesting that in the absence of 12
CatSper3 or CatSper4 protein, sperm initial motility can be maintained when extracellular 13
Ca2+ is not available (Jin et al, 2007). 14
15
After careful evaluation of the null mice, the authors ascribed male infertility to three major 16
factors (Jin et al, 2007). First, a quick loss of motility, significantly reducing the number of 17
Catsper3-null and Catsper4-null spermatozoa that reach the eggs in the ampulla region of the 18
oviduct. Second, the inability of Catsper3-null and Catsper4-null spermatozoa to penetrate 19
the viscous medium suggesting that these mutant sperm cannot progress efficiently in the 20
mucosa of the female reproductive tract, and thus the number of sperm reaching the eggs 21
would be reduced. Third, the lack of hyperactivation compromises the ability of the mutant 22
sperm to generate sufficient forces to penetrate cumulus cells and zona pellucida during 23
fertilization (Jin et al, 2007). This finding suggests that functional CatSper3 and CatSper4 24
9
proteins are required for maintaining the motility and generate forces required for penetrating 1
the oocyte’s outer layers. 2
3
As detailed above, the sperm motility defects in Catsper3_/_ and Catsper4_/_ mice are very 4
similar to those seen in Catsper1_/_ and Catsper2_/_ mice. Thus, lack of any of the four 5
CatSper proteins appears to result in deficiency in hyper-activated motility and quick loss of 6
initial motility in the capacitating conditions. An almost identical phenotype in mice lacking 7
either of the four CatSper channel proteins, their highly homologous domain structure, and 8
their similar localization to the sperm flagellum all suggest that CatSpers 1–4 form a tetramer 9
cation channel, which is required for the development of hyperactivated motility in the 10
female reproductive tract (Jin et al, 2007). Interestingly, Qi et al reported that the four 11
CatSper proteins are indeed associated with each other and form a tetramer (Qi et al, 2007). 12
On the other hand, female CatSpers-null mice have normal fertility, and no differences were 13
observed in litter size and litter intervals compared with wild type females. 14
15
The hormone progesterone released by cumulus cells surrounding the egg is a potent 16
stimulator of human spermatozoa. It attracts spermatozoa towards the egg and helps them 17
penetrate the egg’s protective vestments. Progesterone induces Ca2+ influx into spermatozoa 18
and triggers multiple Ca2+-dependent physiological responses essential for successful 19
fertilization, such as sperm hyper-activation, chemotaxis towards the egg and acrosome 20
reaction. As an ovarian hormone, progesterone acts by regulating gene expression through a 21
well-characterized progesterone nuclear receptor. However, the effect of progesterone upon 22
transcriptionally silent spermatozoa remains unexplained and is believed to be mediated by a 23
specialized, non-genomic membrane progesterone receptor. Progesterone-evoked Ca2+ influx 24
in human sperm does not involve classical regulation of transcription by nuclear receptors. 25
10
Various candidate membrane receptors for progesterone have emerged, including a novel G-1
protein coupled type progestin receptor (mPR) and a single-pass receptor (progesterone-2
receptor membrane component, PGRMC). The pathway downstream of the non-genomic 3
progesterone receptor has been proposed to involve cAMP and cGMP, protein kinase A and 4
G, Ca2+ release from intracellular stores, store-operated Ca2+ channels and cGMP activated 5
channels. 6
7
It is reported that the human CatSper can be further potentiated by prostaglandins, but 8
apparently through a binding site other than that of progesterone. CatSper or a directly 9
associated protein serves as the elusive non-genomic progesterone receptor of sperm and the 10
CatSper associated progesterone receptor is sperm specific and structurally different from the 11
genomic progesterone receptor. It represents a promising target for the development of a new 12
class of non-hormonal contraceptives (Lishko et al, 2011). Weakly voltage-dependent, pH-13
sensitive CatSper is the only constitutively active Ca2+ conductance present in mouse and 14
human spermatozoa as recorded using the whole-cell patch clamp technique. Ca2+ influx 15
through CatSper triggers sperm hyperactivation. CatSper is also ideally positioned to control 16
sperm chemotaxis, because ‘chemotactic turns’ that guide spermatozoa towards the egg 17
depend on asymmetrical flagellar motion triggered by Ca2+ influx into the flagellum. 18
However, CatSper-mediated Ca2+ influx into the flagellum leads to Ca2+ elevation even in the 19
sperm head (probably by causing Ca2+-dependent Ca2+release from the intracellular store 20
located in the sperm neck) and thus can contribute to Ca2+dependent acrosome reaction 21
(Strunker et al, 2011). 22
23
Mutation studies on human subjects have been rare and mutations in only CATSPER2 gene 24
have been linked to non-syndromic human male infertility (Avidan et al, 2003), but other 25
11
human CatSper mutations are likely to be found. Carefully designed studies on human 1
subjects could result in identification of mutations in this or other CatSper genes in infertile 2
individuals. As stated above, CatSpers are required not only for sperm motility and 3
hyperactivation but also for egg penetration, therefore, both asthenozoospermic and 4
normozoospermic infertile individuals are ideal subjects for mutation studies in humans. 5
Because CatSper may cause all three Ca2+ dependent responses triggered by progesterone 6
(hyper-activation, chemotaxis and acrosome reaction). The identification of CatSper channel 7
blockers will greatly facilitate the study of Ca2+ signalling in sperm and help to define further 8
the physiological role of progesterone and CatSper (Strunker et al, 2011). Identification of 9
mutations in CATSPER genes in infertile humans could further potentiate the development of 10
such inhibitors. 11
12
Proton voltage gated ion channel (Hv1) 13
Hv1 exists in the cell membrane as a dimer of identical voltage sensor protomers. The current 14
theory anticipates each sensor to contain its own proton conduction pathway. Hv1 is confined 15
to the principal piece of the sperm flagellum, where it is expressed at unusually high density 16
(Lishko et al, 2010). These tiny cells rely on a proton-shedding pore to speed toward their 17
target. Whole-cell patch-clamp recordings of human spermatozoa reveal high proton 18
conductance. Human spermatozoa are quiescent in the male reproductive system and must 19
undergo activation once introduced into the female reproductive tract. This process is known 20
to require alkalinization of sperm cytoplasm. Robust flagellar Hv1-dependent proton 21
conductance is activated by membrane depolarization, an alkaline extracellular environment, 22
endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 23
allows only outward transport of protons and is therefore dedicated to inducing intracellular 24
alkalinization and activating spermatozoa (Lishko et al, 2010). Since Hv1 and CatSper 25
12
channels are located in the same subcellular domain, proton extrusion via Hv1 channels 1
should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore, the 2
combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of 3
both intracellular pH and Ca2+ required for sperm activation in the female reproductive tract 4
(Lishko et al, 2010). 5
6
Most of the studies finding the importance of gene function in fertility are carried out in 7
animal models such as mice or rat. Since Hv1 current is absent in mouse (Lishko et al, 2010), 8
it is not possible to study the impact of null mutations on fertility using this animal model. 9
Hv1 knockout mouse has been generated; however, as anticipated they have normal fertility 10
(Ramsey et al, 2009). Mutations in the human subjects are likely to result in infertility due to 11
highly significant role of this proton channel in sperm activation and acrosome reaction. 12
However, no study on human subjects has been undertaken till date. Mutations in this gene 13
are not likely to affect sperm count; therefore, infertile individuals having most of the semen 14
parameters in normal range, such as normozoospermic infertile individuals, are best 15
candidates for such studies. The importance of Hv1 in sperm activation makes it an attractive 16
target for controlling male fertility. 17
18
Potassium voltage gated ion channel (SLO3/KCNU1) 19
Among other positively charged ions, K+ ion channels and transporters are essential 20
components of the cellular migration machinery. Non-capacitated murine spermatozoon 21
resting membrane potential is ~−30 mV. After capacitation, sperm cell hyperpolarizes to 22
~−60 mV (Zeng et al, 1995; Arnoult et al, 1999). Using voltage-sensitive fluorophores, the 23
13
capacitation-associated hyperpolarization was ascribed to an increase in K+ permeability 1
(Zeng et al, 1995) and block of epithelial sodium channel (Hernandez-Gonzalez et al, 2006). 2
In whole-sperm patch clamp, a constitutively active, weakly outwardly rectifying K+ current 3
was measured. This potassium current (IKSper) was specifically localized to the principal 4
piece of the sperm flagellum (Navarro et al, 2007). Murine Slo3 (Schreiber et al, 1998) gene 5
is the most likely candidate responsible for IKSper. mSlo3 is testis-specific (expressed in 6
abundance in the mammalian spermatocytes) and its ion channel properties resemble those of 7
IKsper. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm 8
capacitation and/or the acrosome reaction; essential steps in fertilization where changes in 9
both intracellular pH and membrane potential are known to occur (Schreiber et al, 1998). 10
11
Zeng et al, (2011) showed that genetic deletion of Slo3 abolishes all pH-dependent K(+) 12
current at physiological membrane potentials in corpus epididymal sperm. A residual pH-13
dependent outward current (I(Kres)) was observed in Slo3(-/-) sperm at potentials of >0 mV. 14
Differential inhibition of KSper/Slo3 and I(Kres) by clofilium revealed that the amplitude of 15
I(Kres) is similar in both wild-type and Slo3(-/-) sperm. The properties of I(Kres) suggested 16
that it likely represents outward monovalent cation flux through CatSper channels. The 17
authors suggested that KSper/Slo3 may account for essentially all mouse sperm K(+) current 18
and is the sole pH-dependent K(+) conductance in these sperm. With physiological ionic 19
gradients, alkalization depolarized Slo3(-/-) spermatozoa, presumably from CatSper 20
activation, in contrast to Slo3/KSper-mediated hyperpolarization in wild type sperm. It was 21
found that Slo3(-/-) male mice are infertile, but Slo3(-/-) sperm exhibited some fertility in in 22
vitro fertilization assays. Slo3(-/-) sperm exhibited a higher incidence of morphological 23
abnormalities accentuated by hypotonic challenge and also exhibit deficits in motility in the 24
absence of bicarbonate, revealing a role of KSper under unstimulated conditions. Together, 25
14
these results showed that KSper/Slo3 is the primary spermatozoan K(+) current and that 1
KSper may play a critical role in acquisition of normal morphology and sperm motility when 2
faced with hyperosmotic challenges, making Slo3 critical for fertility (Zeng et al, 2011). 3
4
Indispensable role of IKSper in sperm physiology and fertility is apparent from animal model 5
studies. Mutations in the gene encoding this channel are likely to result in sperm with 6
compromised activation and fertility. Therefore, the gene(s) encoding this channel is(are) 7
good candidate(s) for screening in infertile human males. At present Slo3 seems to be the 8
only gene encoding this channel; however, identification of more gene(s) contributing to the 9
formation/activity of this channel could provide more candidate gene(s) for screening in 10
human male infertility. To the best of our knowledge, no such mutation in infertile humans 11
has been reported. Because of its high testis specific expression, pharmacological agents that 12
target human Slo3 channels may be useful in both the study of fertilization as well as in the 13
control or enhancement of fertility. 14
15
Sodium Voltage gated Ion Channel (Nav 1.1 -1.9) 16
Voltage-gated sodium channels (VGSCs) play an essential role in the generation of the rapid 17
depolarization during the initial phase of the action potential in excitable cells. These 18
complex membrane proteins are composed of an α and one or more auxiliary β subunits. The 19
α subunits are large proteins with a high degree of amino acid sequence identity; they contain 20
an ion-conducting aqueous pore and can function without the β subunit as a Na+ channel. 21
Nine different voltage-dependent Na+ channel α subunits have been cloned in mammals, each 22
of which is encoded by a different gene. They can be further characterized by their sensitivity 23
15
to the highly selective blocker tetrodotoxin (TTX). The TTX-sensitive α subunits are 1
inhibited by TTX in the nanomolar range and include SCN1A (also known as Nav1.1), 2
SCN2A (also known as Nav1.2), SCN3A (also known as Nav1.3), SCN4A (also known as 3
Nav1.4), SCN8A (also known as Nav1.6), and SCN9A (also known as Nav1.7). The TTX 4
resistant α subunits are inhibited by TTX in the micromolar range and include SCN5A (also 5
known as Nav1.5), SCN10A (also known as Nav1.8), and SCN11A (also known as Nav1.9). 6
A tenth, related, non voltage dependent α isoform, SCN7A (also known as Nax), has also 7
been reported (Francisco MP et al, 2009). Four different β subunits, SCN1B, SCN2B, 8
SCN3B, and SCN4B (also named β1–4) are currently known (Marian et al, 2009). The roles 9
of the β subunits are less well established, although they appear to modulate the cellular 10
localization, functional expression, kinetics, and voltage-dependence of channel gating. 11
12
Regarding Na+ channels, Hernández-González et al, 2006 reported the involvement of an 13
amiloride-sensitive Na+ channel that may contribute to the regulation of resting sperm 14
membrane potential. The characteristics of these channels match with the family of epithelial 15
Na+ channels. The presence of voltage-dependent Na+ channels in human sperm was first 16
reported by Francisco et al, 2009 and supports a role for these channels in the regulation of 17
mature sperm function. Nav channels could play a more important role in the non-capacitated 18
and in the initial capacitation steps and be inactivated during capacitation, when sperm 19
membrane hyperpolarizes before the acrosome reaction. Null mutations in mice often result 20
in lethal phenotypes making it difficult to study the impact on fertility (Planells-Cases et al, 21
2000). This could be due to more generalized functions of these channels. To the best of our 22
knowledge, no mutation in any of the sodium channel in infertile human subjects has been 23
reported till date. However, mutations in sodium channels in other disorders have been 24
reported (Heron et al., 2010). Before execution of such studies on infertility, it is imperative 25
16
to analyze the sodium channel gene family for relative expression in different organs. Genes 1
with predominant expression in testis/spermatozoa could be the best candidates to begin with; 2
however, it is also possible that mutations in other channels with general distribution 3
selectively impair sperm function without significantly affecting other physiological 4
processes. 5
6
Transient receptor potential (TRP) channel 7
The mammalian TRP ion channel super family consists of 28 members categorized into six 8
sub-families named TRPC (canonical), TRPV (vaniloid), TRPM (melastatine), TRPA 9
(ankyrin), TRPML (mucolipid) and TRPP (polycystins). Many TRP proteins play critical 10
roles in processes such as sensory physiology, vasorelaxation and male fertility (Petra 11
Weissgerber, 2011). The TRPC family has attracted great attention because of their putative 12
roles as store-operated and receptor operated cation channels. In many cells, the emptying of 13
Ca2+ stores generates a gating signal that couples intracellular Ca2+ release to the opening of 14
store-operated channels (SOCs) in the plasma membrane. It has been reported that TRPC 15
channels may act as candidate molecular entities for SOCs which were responsible for the 16
sustained (Ca2+) elevation necessary for normal physiological function (Li et al, 2010 ). 17
18
The expression of TRP and cyclic nucleotide gated (CNG) channels has been detected in both 19
spermatogenic cells and mature sperm (Weyand et al, 1994; Jungnickel et al, 2001; 20
Castellano et al, 2003). They are heterogeneously distributed in these cells, suggesting their 21
participation in distinct functions at particular sperm locations. For example, TRPC2 is 22
present in the head of mouse sperm where it has been proposed to participate in the acrosome 23
17
reaction. Searching for the egg, sperm encounter complex changes in media composition, 1
viscosity and temperature. It is possible that other members of the TRP family are present in 2
sperm to contend with the variety of signalling demands required for fertilization. In 3
particular, TRPM channels are good candidates since they may participate in sensory 4
physiology, both at the cell and whole organism level. TRPM members are responsible for 5
sensing, among other stimuli, temperature, osmolarity, voltage and pH (Venkatachalam and 6
Montell, 2007). Importantly, these channels are often regulated by more than one stimulus 7
and thus regarded as signal integrators. This particular feature is presumably essential for 8
sperm during their adventurous journey towards the egg. However, these are all projections 9
on the basis of functional properties of TRP channels and exact biological functions remain to 10
be understood. 11
12
Few studies have reported the presence of a thermo sensitive channel (TRPM8) in human 13
sperm (De Blas et al, 2009; Martínez-López et al, 2011). This channel might have a role in 14
cellular signalling i.e. thermotaxis and chemotaxis mechanism involved in guiding the sperm 15
during fertilization. Further studies are needed to fully explore the participation of TRPM8 in 16
motility regulation. This is relevant considering recent evidence indicating that mammalian 17
sperm exhibit both thermo and chemotaxis and that they are important for mammalian 18
fertilization. TRPC channels are functionally important in sperm activation (reviewed in 19
Felix, 2005). TRPC1, -2 (only in mice), -3, -4, -6 and TRPP2 are expressed in mammalian 20
sperm (Nilius et al, 2007). TRPC6 localizes to the postacrosomal region of mouse sperm and 21
may be involved in the acrosome reaction. TRPC1 and TRPC3, on the other hand, are 22
confined to the flagellum and might, in concert with other Ca2+-entry channels, be involved in 23
the process of sperm activation as mature sperm are released from the caudal epididymis 24
(Nilius et al, 2007). 25
18
1
Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, RNA messengers 2
for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect 3
immunofuorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the 4
human sperm, and immunogold transmission electron microscopy indicated that TRPC 5
epitopes are mostly associated to the surface of the cells (Castellano et al, 2003). These 6
results provide evidence on proposed function of these ions channels in human sperm 7
motility. The distribution of TRPC channels differs in detail between human and mouse, but 8
TRPC1, -3, -4, and -6 have all been detected within the flagellum, suggestive of a role in 9
motility (Castellano et al, 2003). Pharmacological suppression of TRPC channel activity 10
causes impairments in the motility of human sperm; however, the agents employed exhibit 11
rather poor pharmacological selectivity (Castellano et al, 2003). TRPC3 also plays a role in 12
fluid formation in the epididymis (Cheung et al, 2005). While transient increases in [Ca2+]i 13
via T-type Ca2+ channels might be necessary to trigger sperm capacitation, the acrosome 14
reaction requires sustained Ca2+ entry, which may occur via TRPC channels. TRPC channels 15
do not seem to be crucial for hyperactivation, which requires the sperm-specific channels 16
CatSper1 and CatSper2 (Darszon et al, 2001; Trevion et al, 2001; Clapham and Garbers, 17
2005). The exact contribution of this channel to Ca2+ transport needs to be worked out in 18
detail. 19
20
Targeted disruption of a trpp2 homolog in Drosophila, pkd2, which is expressed in the head 21
and the tail of the sperm, resulted in male infertility without an effect on spermatogenesis. 22
Instead, the mutant sperms, although motile, were unable to reach the sperm storage organ in 23
the females (seminal receptacles and spermathecea) (Gao et al, 2003). A similar deficit in 24
19
directional movement is observed following targeted disruption of a second Drosophlia trpp2 1
homolog termed, most appropriately, "almost there" (amo) that localizes to the distal tip of 2
the flagellum (Watnik et al, 2003). It is intriguing that sensory functions of TRPP2 homologs 3
are conserved across evolution in both motile (flagellum) and non-motile (monocilium) 4
axonemal-containing structures. Therefore, mutation studies in human male infertility are 5
likely to result in identification of new causes of infertility, particularly in the cases with 6
unexplained infertility having all other normal semen parameters. However, first infertile 7
man with such mutation(s) is yet to be identified. 8
9
Cystic fibrosis trans-membrane conductance regulator (CFTR) 10
Cystic fibrosis (CF) is a common hereditary disease caused by mutations of the gene 11
encoding cystic fibrosis transm-+embrane conductance regulator (CFTR), a cAMP-activated 12
anion channel, with clinical manifestations of progressive lung disease, pancreatic 13
insufficiency, and infertility in both sexes (Quinton, 1990). Infertility in CF male patients is 14
mostly due to congenital bilateral absence of the vas deferens (CBAVD) and obstructive 15
azoospermia. Interestingly, higher prevalence of CFTR mutations in otherwise healthy men 16
presenting with reduced sperm quality compared with controls has been reported (van der 17
Ven et al, 1996). Another study showed that CFTR heterozygous form is 2 fold higher in 18
infertile individuals in comparison to general population (Schulz et al, 2006). However, the 19
exact role of CFTR in this context remained obscure till very recent. 20
21
As mentioned elsewhere in this article, capacitation is known to be associated with elevation 22
of intracellular pH (Meizel and Deamer, 1978) and hyperpolarization of the sperm plasma 23
membrane (Zeng et al., 1995). These events have been shown to depend on extracellular 24
20
HCO3- (Demarco et al., 2003). The regulatory role of HCO3- in sperm capacitation has been 1
attributed to its direct activation of a soluble form of adenylyl cyclase (Chen et al, 2000) that 2
in turn activates cAMP production and various downstream cellular events, such as protein 3
tyrosine phosphorylation, leading to capacitation (Visconti et al., 2002). Despite the 4
mounting evidence indicating the key importance of HCO3- in sperm capacitation, the 5
transporting mechanism responsible for the entry of HCO3- into sperm remained poorly 6
understood till very recent. 7
8
CFTR is a channel protein known to conduct both Cl- and HCO3-, defects of which due to its 9
gene mutations cause CF. CFTR expression had been shown in the testis of rodents (Trezise 10
et al., 1993) and rat germ cells (Gong et al, 2001), its expression and function in mature 11
sperm was demonstrated only recently (Xu et al., 2007). The authors examined the expression 12
of CFTR in both human and mouse sperm, using immunofluorescence staining and Western 13
blot analysis. Using confocal imaging, CFTR was immunolocalized to the equatorial segment 14
of human and mouse sperm. Western blot analysis using a mouse antihuman CFTR 15
monoclonal antibody also revealed the presence of CFTR in mouse sperm (Xu et al, 2007). 16
The authors conducted further experiments to find if CFTR is involved in mediating the 17
HCO3�entry important for sperm capacitation. The use of CFTR inhibitor reduced the 18
number of capacitated sperm, suggesting its role in sperm capacitation. Experiments using 19
CFTR inhibitors and anti CFTR antibody also revealed significant role of this transporter in 20
sperm intracellular pH increase by mediating HCO3- entry during capacitation. Sperm from 21
the heterozygous Cftr+/-� mice showed reduced ability to undergo capacitation compared 22
with those from wild-type littermates (C57BL/6J background). The results showed that after 23
2-h incubation in the capacitation-inducing medium, the percentage of capacitated sperm of 24
21
the heterozygous mutant mice was significantly lower than that of the wild-type control. 1
Further investigation on this model revealed a defect in HCO3- transport (Xu et al, 2007). 2
3
To further demonstrate the role of this transported in fertility, the authors performed in vitro 4
fertilization to compare the fertilizing capacity of heterozygous Cftr+/- sperm to that of Cftr +/+ 5
sperm. The results showed that the percentage of fertilized eggs when incubated with Cftr+/- 6
sperm (13 of 73, 16%) was significantly lower than that with Cftr+/+ sperm (28 of 60, 48.5%). 7
The impaired fertilizing capacity in Cftr+/- sperm could also be evidenced by their reduced 8
binding and penetration of zona pellucida-free eggs compared with the wild-type sperm. To 9
further demonstrate the role of CFTR in male fertility in a physiological context, the authors 10
examined the fertility rate of male Cftr+/- mice through natural mating. Cftr+/+ female mice 11
housed overnight with Cftr+/- males showed normal number of vaginal plugs the following 12
morning. However, only five of the nine Cftr+/- males tested had offspring, compared with 13
100% of the age matched wild-type males having offspring, and the averaged litter size in 14
females mated with the Cftr+/- males was also reduced. 15
16
The authors also reported significant reduction in sperm motility with compromised forward 17
movement parameters in Cftr+/- mice compared with wild-type control. These findings are 18
also in line with a role of CFTR in transporting HCO3-, because HCO3- has also been linked 19
to sperm motility (Abaigar et al., 1999; Holt and Harrison, 2002). These findings of the 20
previously unsuspected role of CFTR in sperm function may unravel the mysteries of many 21
unexplained cases of male infertility, because ~1,200 mutations in CFTR have been identified 22
since its discovery with various defects (Rowe et al, 2005). These mutations may affect 23
CFTR function to different extents, which may explain the observation that certain CFTR 24
mutations are associated with reduced sperm quality in men who do not present CF 25
22
phenotype. CFTR may also play different roles in sperm function other than HCO3- transport 1
for sperm capacitation, because CFTR is also known to regulate an array of other proteins 2
and cellular processes. Together with the reported involvement of CFTR in HCO3- secretion 3
by the female reproductive tract (Wang et al, 2003), the present findings of CFTR 4
involvement in sperm capacitation indicates that CFTR may have crucial role in reproduction 5
in both sexes. 6
7
Other calcium (cation) channels 8
Intracellular pH and concentration of Ca2+ is increased during capacitation. As detailed 9
above, the role of CatSpers in sperm hyperactivation is well established. Many other Ca2+-10
permeable channels, including classical voltage-gated Ca2+ (CaV), TRP and CNG have been 11
proposed to participate in regulation of intracellular Ca2+ concentration (Darszon et al, 2006). 12
The importance of these calcium channels is highlighted by the presence of Cav, TRPC2 and 13
CNGA3 channels on mouse spermatozoa (Carlson et al, 2003; Darszon et al, 2006). Some of 14
these channels do not seem indispensable as the mice carrying null mutations for CaV1.3 15
(Platzer et al, 2000), CaV2.2 (Ino et al, 2001), CaV2.3 (Saegusa et al, 2000), CaV3.1 (Kim et 16
al, 2001) and CaV3.2 (Chen et al, 2003), TRPC2 (Stowers et al, 2002) and CNGA3 (Biel et 17
al, 1999) have normal fertility. The experiments to find importance of other calcium channels 18
in spermatogenesis and fertility are compounded by the fact that mutants of cetain channels 19
are lethal. For example, CaV1.2 null mutant (Seisenberger et al, 2000) is embryonic lethal and 20
CaV2.1 null pups die ~3-4 weeks after birth (Jun et al, 1999) before fertility can be examined. 21
Nevertheless, their importance in sperm fertility remains to be established. 22
23
23
CaV channels consist of a pore-forming α1 subunit that determines the ion selectivity, a β 1
subunit (an intracellular protein), a single TM-spanning α2/δ subunit, and a multiple 2
membrane spanning c subunit (Catterall et al, 1999). Similarly, voltage-gated Na+ (NaV) 3
channel are composed of the pore forming α subunit and the single TM-spanning β subunits. 4
The auxiliary subunits have fundamental roles in the formation and localization of the 5
channels, as their presence influences the biophysical properties of the channels reconstituted 6
in heterologous expression systems (Arikaath and Campbell, 2003). However, these subunits 7
are also essential for the channel function in vivo, as mutations in CaV b (Barclay et al, 2001) 8
or α 2/δ subunits lead to severe disorders or lethality. 9
10
Conclusion and future directions 11
The importance of ion channels in spermatogenesis and sperm maturation is emphasized by 12
the expression of several ion channels in the testis, epididymis and ejaculated sperm. Most of 13
these ion channels are essential for sperm motility, sperm activation, acrosome reaction and 14
journey towards the egg for fertilization. The role of CatSper genes in sperm hyperactivation 15
has been well established, Hv1 and K+ channels are essential for changes in intracellular pH 16
and membrane potential and sodium channels are required for sperm activation. TRPC family 17
members serve the function of chemo and thermo sensing, guiding sperm to its ultimate 18
target, i.e. egg. Therefore, the importance of ion channels in sperm physiology has been 19
established beyond doubt. However, functional specific roles of each member of particular 20
channels remain unexplored. For example, Ca2+ influx is carried out by several different ion 21
channels; however, contribution of each member and the exact process it influences, remains 22
to be established. Null mouse models for several ion channels have been generated and most 23
24
of them resulted in impaired fertility either due to loss of motility, hyperactivity or the ability 1
to fertilize the egg despite other semen parameters being normal. 2
3
Despite the indispensable role of the ion channels as evidenced by animal models, mutation 4
screening studies in infertile humans have been obscure to the extent that mutation(s) only in 5
CatSper 2 gene have been reported. Availability of no report on other genes shows absence of 6
any study screening these genes. The molecular signalling of the ion channels during 7
defective and normal spermatogenesis, maturation, capacitation and acrosomal reaction, need 8
to be explored by studying sperm from well characterized fertile and infertile individuals. The 9
understanding of differences in the function would further help identify etiology of male 10
infertility in patients with undefined etiology. For example, function of the ion channels in 11
spermatogonia would be different if compared to the functional characteristic of the same in 12
the ejaculated sperm. More research is required to be infused in understanding these 13
differences. Further, the thermo- and chemo-sensing by sperm is now known to guide it 14
towards ovum. For example, the role of TRPC family in such signalling has been 15
emphasized; however, exact role of each member and its ligand/signalling molecule remains 16
to be identified. Identification of the role of ion channels in such signalling would help 17
further understand sperm physiology and identify opportunities for achieving fertility or 18
contraception. 19
20
In essence, the role of ion channels in sperm physiology and male fertility is well established 21
and appears to be indispensable. However, molecular characterizations and screening of the 22
ion channels in infertile individuals has to confirm its role in male infertility as suggested by 23
animal studies. Asthenozoospermic and normozoospermic infertile individuals could be the 24
25
best candidates for such screening due to the likely sperm function defects in these 1
individuals. It may also open novel drug targeting strategies to design a functional male 2
contraceptive. For example, the inhibitory effect of genistein on T-type calcium channels was 3
associated with a hyperpolarizing shift resulting in significant inhibition of sperm acrosome 4
reaction and its binding to zona pellucida. Using transfected HEK293 cells system, it was 5
found that genistein inhibited only Cav3.1 and Cav3.2 and not Cav3.3. Since T-type calcium 6
channels are the key components in the male reproduction, such as in acrosome reaction and 7
sperm motility, this may explain anti-fertility effects of genistein (Tao et al, 2009). Further 8
studies on ion channels have a wide scope and plethora of information is waiting to be 9
uncovered. 10
11
As mentioned in the beginning of this article, a highly specific process such as fertilization 12
has to be strictly directed and target oriented. Sperm cannot afford to wait for a chance union 13
with the oocyte. Specific mechanisms guiding the sperm to the occyte must exist to increase 14
the probability of fertilization which is imperative for achieving maximum fecundity under 15
given conditions. Identification of the mechanisms such as thermo- and chemo-taxis has 16
emphasized on directed movements of sperm towards the oocyte. Similarly, a specific 17
sequence of the action of the ion channels is highly likely to guide the sperm and initiate 18
specific changes required at each stage of transport ultimately making the sperm capable of 19
fertilizing the oocyte. There may be functional overlap between different ion channels but a 20
highly coordinated and orchestrated action is likely to exist which ensures that no mistake is 21
committed in the execution of the biologically most important task. Nature has provided us 22
the opportunity in the form of a non-fertile sperm being ejaculated in the female, which needs 23
to gain fertility before it can be biologically meaningful. Understanding the ion transport in 24
26
directing the sperm and determining its fertility could provide us the most valuable key to 1
develop a reversible male contraceptive and/or treat infertility. 2
3
27
Acknowledgement 1
The authors are thankful to the Ministry of Health and Family Welfare (MOH & FW) for 2
financial assistance. 3
1
References
Abaigar T, Holt WV, Harrison RA, del Barrio G. Sperm subpopulations in boar (Sus scrofa)
and gazelle (Gazella dama mhorr) semen as revealed by pattern analysis of
computer-assisted motility assessments. Biol Reprod. 1999;60:32-41.
Anne E. Carlson, Timothy A. Quill, Ruth E. Westenbroek, Sonya M. Schuh, Bertil Hille,
Donner F. Babcock. Identical Phenotypes of CatSper1 and CatSper2 Null Sperm. J
Bio Chem 2005;280:32238–32244.
Arikkath J, Campbell KP. Auxiliary subunits: essential components of the voltage-gated
calcium channel complex. Curr Opin Neurobiol 2003; 13:298-307.
Arnoult C, Kazam IG, Visconti GS, Villaz M, Floman HM. Control of the low voltage
activated calcium channel of mouse sperm by egg ZP3 and by membrane
hyperpolarization during capacitation. Proc Natl Acad Sci USA. 1999;96:6757-
6762.
Avidan N, Tamary H, Dgany O, Cattan D, Ariente A, Thulliez M, Borot N, Moati L,
Barthelme A, Shalmon L. et al. .CATSPER2, a human autosomal nonsyndromic
male infertility gene. Eur J Hum Genet. 2003;11:497-502.
Babcock DF. Wrath of the wraiths of CatSper3 and CatSper4. Proc Natl Acad Sci USA.
2007;104:1107–1108.
Bahat A, Tur-Kaspa I, Gakamsky A, Giojalas LC, Breitbart H, Eisenbach M. Thermotaxis of
mammalian sperm cells: a potential navigation mechanism in the female genital
tract. Nat Med. 2003 Feb;9(2):149-50. No abstract available.
2
Barclay J, Balaguero N, Mione M, Ackerman SL, Letts VA, Brodbeck J, Canti C, Meir A,
Page KM, Kusumi K, Perez-Reyes E, Lander ES, et al. Ducky mouse phenotype of
epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and
decreased calcium channel current in cerebellar Purkinje cells. J Neurosci. 2001;
21:6095-6104.
Biel M, Seeliger M, Pfeifer A, Kohler K, Gerstner A, Ludwig A, Jaissle G, Fauser S, Zrenner
E, Hofmann F. Selective loss of cone function in mice lacking the cyclic
nucleotide-gated channel CNG3. Proc Natl Acad Sci USA.1999;96:7553-7557.
Carlson AE, Westenbroek RE, Quill T, Ren D, Clapham DE, Hille B, DL, Babcock DF.
CatSper1 required for evoked Ca2þ entry and control of flagellar function in
sperm. Proc Natl Acad Sci USA. 2003;100:14864-14868.
Castellano LE, Trevino CL, Rodriguez D, Serrano CJ, Pacheco J, Tsutsumi V, Felix R,
Daeszon A. Transient receptor potential (TRPC) channels in human sperm:
expression, cellular localization and involvement in the regulation of flagellar
motility. FEBS Lett. 2003;541:69-74.
Catterall WA. Structure and function of voltage-gated ion channels. Annu Rev Biochem
1995; 64:493–531.
Chen CC, Lamping KG, NunoDW,Barresi R, Prouty SJ, Lavoie JL, Cribbs LL, England SK,
Sigmund CD, Weiss RM, Williamson RA, Hill JA, Campbell KP. Abnormal
coronary function in mice deficient in alpha1H T-type Ca2 channels. Science.
2003;302:1416-1418.
3
Chen Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, Levin LR, Buck J. Soluble adenylyl
cyclase as an evolutionarily conserved bicarbonate sensor. Science. 2000;289:625-
628.
Cheung KH, Leung GP, Leung MC, Shum WW, Zhou WL, Wong PY. Cell-cell interaction
underlies formation of fluid in the male reproductive tract of the rat. J Gen Physiol.
2005;125:443-454.
Clapham DE, Garbers DL. International Union of Pharmacology. L. Nomenclature and
structure-function relationships of CatSper and two-pore channels. Pharmacol Rev.
2005;57:451–454.
Collingridge GL, Olsen RW, Peters J, Spedding M. A nomenclature for ligand-gated ion
channels. Neuropharmacology. 2009;56:2-5.
Darszon A, Acevedo JJ, Galindo BE, Hernandez-Gonzalez EO, Nishigaki T, Trevino CL,
Wood C, Beltran C. Sperm channel diversity and functional multiplicity.
Reproduction. 2006;131:977-988.
Darszon A, Beltran C, Felix R, Nishigaki T, Trevino CL. Ion transport in sperm signaling.
Dev Biol. 2001;240:1-14.
Darszon A, Labarca P, Nishigaki T, Espinosa F. Ion channels in sperm physiology. Physiol
Rev. 1999;79:481-510.
De Blas GA, Darszon A, Ocampo AY, Serrano CJ, Castellano LE, Hernández-González EO,
Chirinos M, Larrea F, Beltrán C, Treviño CL. TRPM8, a versatile channel in
human sperm. PLoS One. 2009;4:e6095.
4
Demarco IA, Espinosa F, Edwards J, Sosnik J, De La Vega-Beltran JL, Hockensmith JW,
Kopf GS, Darszon A, Visconti PE. Involvement of a Na+/HCO-3 cotransporter in
mouse sperm capacitation. J Biol Chem. 2003;278:7001-7009.
Eisenbach M. Mammalian sperm chemotaxis and its association with capacitation. Dev
Genet. 1999;25:87-94.
Felix R. Molecular physiology and pathology of Ca2_-conducting channels in the plasma
membrane of mammalian sperm. Reproduction. 2005;129:251-262.
Francisco MP, Cristina GR, Manuel FS, Manuel GC, Antonio CR, and Luz C. Molecular and
functional characterization of voltage-gated sodium channels in human sperm.
Reprod Biol Endocrinol. 2009;7:71.
Gao Z, Ruden DM, Lu X. PKD2 cation channel is required for directional sperm movement
and male fertility. Curr Biol. 2003;13:2175-2178.
Gong XD, Li JC, Cheung KH, Leung GP, Chew SB, Wong PY. Expression of the cystic
fibrosis transmembrane conductance regulator in rat spermatids: implication for the
site of action of antispermatogenic agents. Mol Hum Reprod. 2001;7:705-713.
Haikun Wang, Jin Liu, Kwang-Hyun Cho,3 and Dejian Ren. A Novel, Single,
Transmembrane Protein CATSPERG Is Associated with CATSPER1 Channel
Protein. Biol Reprod 2009;81:539-544.
Hanson FW, Overstreet JW. The interaction of human spermatozoa with cervical mucus in
vivo. Am J Obstet Gynecol. 1981 May 15;140(2):173-178.
Harrison RA, Gadella BM. Bicarbonate-induced membrane processing in sperm capacitation.
Theriogenology. 2005;63:342-351.
5
Hernández-González EO, Sosnik J, Edwards J, Acevedo JJ, Mendoza-Lujambio I, López
González I, Demarco I, Wertheimer E, Darszon A, Visconti PE. Sodium and
epithelial sodium channels participate in the regulation of the capacitation-
associated hyperpolarization in mouse sperm. J Biol Chem. 2006;281:5623-3563.
Heron SE, Scheffer IE, Iona X, Zuberi SM, Birch R, McMahon JM, Bruce CM, Berkovic SF,
Mulley JC. De novo SCN1A mutations in Dravet syndrome and related epileptic
encephalopathies are largely of paternal origin. J Med Genet. 2010;47:137-141.
Ho HC, Suarez SS. Hyperactivation of mammalian spermatozoa: function and regulation.
Reproduction. 2001;122:519–526.
Holt WV, Harrison RA. Bicarbonate stimulation of boar sperm motility via a protein kinase
A-dependent pathway: between-cell and between-ejaculate differences are not due
to deficiencies in protein kinase A activation. J Androl. 2002;23:557-565.
Ino, M., T. Yoshinaga, M. Wakamori, N. Miyamoto, E. Takahashi et al. Functional disorders
of the sympathetic nervous systeminmice lacking the 1B subunit (Cav 2.2) ofN-
type calcium channels. Proc Natl Acad Sci USA. 2001;98:5323-5328.
Jean-Ju Chung, Betsy Navarro, Grigory Krapivinsky, Luba Krapivinsky, David E. Clapham.
Nat Commun. 2011;2:153.
Jin J, Jin N, Zheng H, Ro S, Tafolla D, Sanders KM, Yan W. Catsper3 and Catsper4 are
essential for sperm hyperactivated motility and male fertility in the mouse. Biol
Reprod. 2007;77:37-44.
Jin JL, O'doherty AM, Wang S, Zheng H, Sanders KM, Yan W. Catsper3 and catsper4
encode cation channel-like proteins exclusively expressed in the testis. Biol
Reprod. 2005;73:1235-1242.
6
Jun K, Piedras-Renteria ES, Smith SM, Wheeler DB, Lee SB, Lee TG, Chin, H, Adams ME,
Scheller RH, Tsien RW. et al. Ablation of P/Q-type Ca(2+) channel currents,
altered synaptic transmission, and progressive ataxia in mice lacking the
alpha(1A)-subunit. Proc Natl Acad Sci USA.1999;96:15245-5250.
Jungnickel MK, Marrero H, Birnbaumer L, Lemos JR Floeman HM.Trp2 regulates entry of
Ca2+ into mouse sperm triggered by egg ZP3.Nat Cell Biol. 2001;3:499-502.
Katz DF, Slade DA, Nakajima ST. Analysis of pre-ovulatory changes in cervical mucus
hydration and sperm penetrability. Adv Contracept. 1997;13:143-151.
Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, Shin HS. Lack of the
burst firing of thalamocortical relay neurons and resistance to absence seizures in
mice lacking alpha(1G) T-type Ca(2+) channels. Neuron. 2001;31:35-45.
Li HG, ding XF, Liao AH, Kong XB, Xiong CL. Expression of CatSper family transcripts in
the mouse testis during post-natal development and human ejaculated spermatozoa:
relationship to sperm motility. Mol Hum Reprod. 2007;13:299-306.
Li S, Wang X, Ye H, Gao W, Pu X and Yang Z. Distribution profiles of transient receptor
potential melastatin- and vanilloid-related channels in rat spermatogenic cells and
sperm. Mol Biol Rep. 2010;37:3:1287-1293.
Lishko PV, Kirichok Y. The role of Hv1 and CatSper channels in sperm activation. J Physiol.
2010;588:4667-4672.
Lobley A, Pierron V, Reynolds L, Michalovich D. Identification of human and mouse
CatSper3 and CatSper4 genes: characterisation of a common interaction domain
and evidence for expression in testis. Reprod Biol Endocrinol. 2003;1:53.
7
Marian S, Francisco M. Pinto, Susan W, Cristina G. Cintado, Pedro N, Helmut Buschmann,
Luz Candenas. Functional and molecular characterization of voltage-gated sodium
channels in uteri from non-pregnant rats. Reprod Biol Endocrin. 2009;7:71-80.
Marquez B, Ignotz G, Suarez SS. Contributions of extracellular and intracellular Ca(2þ) to
regulation of sperm motility: release of intracellular stores can hyperactivate
CatSper1 and CatSper2 null sperm. Dev Biol. 2007;303:214-221.
Martínez-López P, Treviño CL, de la Vega-Beltrán JL, De Blas G, Monroy E, Beltrán C,
Orta G, Gibbs GM, O'Bryan MK, Darszon A.TRPM8 in mouse sperm detects
temperature changes and may influence the acrosome reaction. J Cell Physiol.
2011;226:1620-1631.
Meizel S, Deamer DW. The pH of the hamster sperm acrosome. J Histochem Cytochem.
1978;26:98-105.
Morisawa M. Cell signalling mechanisms for sperm motility. Zool Sci. 1994;11:647–662.
Navarro B, Kirichok Y, Chung JJ, Clapham DE. Ion channels that control fertility in
mammalian spermatozoa. Int J Dev Biol. 2008;52:607-613.
Navarro B, Kirichok Y, Clapham DE. KSper a pH- sensitive K+ current that controls sperm
membrane potential. Proc Natl Acad Sci USA. 2007;104:7688-7692.
Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in
disease. Physiol Rev. 2007;87:165-217.
Petra Weissgerber, Ulrich Kriebs, Volodymyr Tsvilovskyy, Jenny Olausson, Oliver Kretz,
Christof Stoerger, Rudi Vennekens, Ulrich Wissenbach, Ralf Middendorff, Veit
8
Flockerzi, Marc Freichel. Male Fertility Depends on Ca2+ Absorption by TRPV6
in Epididymal Epithelia. Sci Signal. 2011;4:ra27.
Planells-Cases R, Caprini M, Zhang J, Rockenstein EM, Rivera RR, Murre C, Masliah E,
Montal M. Neuronal death and perinantal lethality in voltage- gated sodium
channel alpha (II)deficient mice. Biophys J. 2000;78:2878-2891.
Platzer J., Engel J., Schrott-Fischer A., Stephan K., Bova S., Chen H., Zheng H. and
Striessnig J. Congenital deafness and sinoatrial node dysfunction in mice lacking
class D L-type Ca2+ channels. Cell. 2000;102:89–97.
Qi H, Moran MM, Navarro B, Chong JA, Krapivinsky G, Krapivinsky L, Kirichok Y,
Ramsey IS, Quill TA, Clapham DE. All four CatSper ion channel proteins are
required for male fertility and sperm cell hyperactivated motility. Proc Natl Acad
Sci USA. 2007;104:1219-1223.
Quill TA, Ren D, Clapham DE, Garbers DL. A voltage-gated ion channel expressed
specifically in spermatozoa. Proc Natl Acas Sci USA. 2001;98:12527-12531.
Quill TA, Sugden SA, Rossi KL, Doolittle LK, Hammer RE, Garbers DL. Hyperactivated
sperm motility driven by CatSper2 is required for fertilization. Proc Natl Acad Sci
USA. 2003;100:14869-14874.
Quinton PM. Cystic fibrosis: a disease in electrolyte transport. FASEB J. 1990;4:2709-2717.
Ramsey IS, Ruchti E, Kaczmarek JS, Clapham DE. Hv1 proton channels are required for
high-level NADPH oxidase-dependent superoxide production during the phagocyte
respiratory burst. Proc Natl Acad Sci USA. 2009;106:7642-7647.
Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q, Tilly JL, Clapham DE. A sperm ion
channel required for sperm motility and male fertility. Nature. 2001;413:603-609.
9
Rodriguez-Martinez H. Role of the oviduct in sperm capacitation. Theriogenology. 2007;68
Suppl 1:S138-46.
Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med. 2005;352:1992-2001.
Saegusa H, Kurihara T, Zong S, Minowa O, Kazuno A, Han W, Matsuda Y, Yamanaka H,
Osanai M, Noda T et al. Altered pain responses in mice lacking alpha 1e subunit of
the voltage-dependent Ca2þ channel. Proc Natl Acad Sci USA. 2000;97:6132–
6137.
Sarah E Heron, Ingrid E Scheffer, Xenia Iona, Sameer M Zuberi, Rachael Birch, Jacinta M
McMahon, Carla M Bruce, Samuel F Berkovic, John C MulleyDe novo SCN1A
mutations in Dravet syndrome and related epileptic encephalopathies are largely of
paternal origin. J Med Genet 2010;47:137-141.
Schreiber M, Wei A, Yuan A, Gaut J, Saito M, Salkoff L. Slo3, a novel pH-sensitive K+
channel from mammalian spermatocytes. J Biol Chem. 1998;273:3509-3516.
Schultz N, Hamra FK, Garbers DL. A multitude of genes expressed solely in meiotic or
postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc Natl
Acad Sci USA. 2003;100:12201-12206.
Schulz S, Jakubiczka S, Kropf S, Nickel I, Muschke P, Kleinstein J. Increased frequency of
cystic fibrosis transmembrane conductance regulator gene mutations in infertile
males. Fertil Steril. 2006;85:135-138.
Seisenberger C, Specht V, Welling A, Platzer J, Pfeifer A, Kuhbandner S, Striessnig J,
Klugbauer N, Feil R, Hofmann F. Functional embryonic cardiomyocytes after
disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse. J
Biol Chem. 2000;275:39193-3919.
10
Sobrero AJ, Macleod J. The immediate postcoital test. Fertil Steril. 1962;13:184-189.
Stowers L, HolyTE, Meister M, Dulac C, Koentges G. Loss of sex discrimination and male-
male aggression in mice deficient for TRP2. Science. 2002;295:1493-1500.
Suarez SS, Ho HC. Hyperactivation of mammalian sperm. Cell Mol Biol (Noisy-le-grand)
2003;49:351–356.
Suarez SS, Pacey AA. Sperm transport in the female reproductive tract. Hum Reprod Update.
2006;12:23-37.
Tao J, Zhang Y, Li S, Sun W, Soong TW. Tyrosine kinase-independent inhibition by
genistein on spermatogenic T-type calcium channels attenuates mouse sperm
motility and acrosome reaction. Cell Calcium Tao J. 2009;45:133-143.
Timo Stru¨nker, Normann Goodwin, Christoph Brenker, Nachiket D. Kashikar, Ingo
Weyand, Reinhard Seifert, U. Benjamin Kaupp. The CatSper channel mediates
progesterone-induced Ca21 influx in human sperm. Nature 2011;471:382-386.
Trevino CL, Serrano CJ, Beltran C, Felix R, Darszon A. Identification of mouse trp
homologs and lipid rafts from spermatogenic cells and sperm. FEBS Lett.
2001;509:119–125.
Trezise AE, Linder CC, Grieger D, Thompson EW, Meunier H, Griswold MD, Buchwald M.
CFTR expression is regulated during both the cycle of the seminiferous epithelium
and the oestrous cycle of rodents. Nat Genet. 1993;3:157-164.
van der Ven K, Messer L, van der Ven H, Jeyendran RS, Ober C. Cystic fibrosis mutation
screening in healthy men with reduced sperm quality. Hum Reprod. 1996;11:513-
517.
11
Venkatachalam K, Montell C. TRP channels. Annu Rev Biochem. 2007;76:387-417.
Visconti PE, Westbrook VA, Chertihin O, Demarco I, Sleight S, Diekman AB. Novel
signaling pathways involved in sperm acquisition of fertilizing capacity. J Reprod
Immunol. 2002;53:133-150.
Wang XF, Zhou CX, Shi QX, Yuan YY, Yu MK, Ajonuma LC, Ho LS, Lo PS, Tsang LL,
Liu Y, Lam SY, Chan LN, Zhao WC, Chung YW, Chan HC. Involvement of
CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm. Nat
Cell Biol. 2003;5:902-906.
Watnick TJ, Jin Y, Matunis E, Kernan MJ, Montell C. A flagellar polycystin-2 homolog
required for male fertility in Drosophila. Curr Biol. 2003;13:2179–2184.
Weyand I, Godde M, Frings S, Weiner J, Müller F, Altenhofen W, Hatt H, Kaupp UB
Cloning and functional expression of a cyclic-nucleotide-gated channel from
mammalian sperm. Nature. 1994; 368:859-863.
Zeng XH, Yang C, Kim ST, Lingle CJ, Xia XM. Deletion of the Slo3 gene abolishes
alkalization-activated K+ current in mouse spermatozoa. Proc Natl Acad Sci USA.
2011;108:5879-5884.
Zeng Y, Clark EN, Florman HM. Sperm membrane potential: hyperpolarization during
capacitation regulates zona pellucida-dependent acrosomal secretion. Dev
Biol.1995;171:554-563.
Zeng Y, Clark EN, Florman HM. Sperm membrane potential: hyperpolarization during
capacitation regulates zona pellucida-dependent acrosomal secretion. Dev Biol.
1995;171:554-563.
12
Zhang D, Chen J, Saraf A, Cassar S, Han P, Rogers JC, Brioni JD, Sullivan JP,
Gopalakrishnan M. Association of Catsper1 or 2 with Ca(v)3.3 leads to
suppression of T-type calcium channel activity. J Biol Chem. 2006;281:22332–
22341.