http://informahealthcare.com/aanISSN: 1939-6368 (print), 1939-6376 (electronic)

Syst Biol Reprod Med, Early Online: 1–7! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2013.869273

REVIEW AND HYPOTHESIS

Motility, viability, and calcium in the sperm cells

Jorge Parodi

Laboratorio de Fisiologıa de la Reproduccion, Escuela de Medicina Veterinaria, Nucleo de Investigacion en Produccion Alimentaria, Facultad de

Recursos Naturales, Universidad Catolica de Temuco, Temuco, Chile

Abstract

Sperm cells are complicated in vitro models. Their viability is limited, and physiology iscomplex. The study of their properties is of great application in the animal production asviable and functional gametes are essential. It has been shown that the decrease of spermcell viability parallels an increase of the reactive oxygen species (ROS). Reactive oxygenspecies is secondary to normal metabolic processes of the cell-like flagellar movement.There is evidence of strategies that reduce ROS levels by using exogenous or endogenousantioxidants with the intention that seminal plasma protects the sperm cells andincreases viability. Perhaps viability can increase by reducing that flagellar movementwhich is regulated by calcium. The phenomenon has not been fully characterized, but it isestablished that in certain mammalian models, the entrance of calcium via specific channelssuch as CATsper or voltage-dependent channels, signals flagellar movement. Previousreports have indicated that a change in the concentration of calcium or if the temperatureis altered, the function of mammal sperm cells is reduced or blocked and viabilityprolonged. Fish sperm can remain immobile for several weeks but when activated thenumber of mobile and viable sperm is reduced at a faster rate. However, if the cells are notmobilized the semen can be preserved for longer periods. As presented in this paper, thissupports the notion that by modulating calcium channels to reduce motility the viability ofthese cells can increase.

Abbreviations: ROS: reactive oxygen species; ZP: zona pellucid; AR: acrosome reaction; DF:disinhibit factor; TEA: tetraethylammonium chloride; CAVs: calcium voltage channels; CatSper:cationic sperm

Keywords

Calcium, motility, sperm

History

Received 4 July 2013Revised 2 October 2013Accepted 4 October 2013Published online 13 December 2013

Sperm capacitation

Fertilization is a unique and amazing process involving

two morphologically distinct cells, the sperm and the oocyte,

which are recognized and fused together. This process begins

when the sperm starts to penetrate the oocyte envelope and

plasma membrane and ends in the exchange of maternal and

paternal chromosomes, forming the zygote [Patrat et al.

2006]. The sperm must undergo functional changes following

its genesis and subsequent maturation in the epididymis.

Only sperm that have become capacitated can recognize and

bind to the zona pellucida (ZP). The interaction between the

sperm and the ZP initiates a signal transduction process

resulting in exocytosis of the acrosomal contents during the

acrosome reaction (AR) [Breitbart 2003; Rossato et al. 2001].

However, this is only a general picture of the AR phenom-

enon, and some reports have suggested that an intact ZP is

not sufficient to induce acrosomal exocytosis [Baibakov

et al. 2007]. Furthermore, according to the work of

Dr. Yanagimachi’s group, some mouse sperm passing through

the cumulus layers are already undergoing or have completed

the acrosome reaction [Knobil and Neill 1994]. In shrews, the

acrosome reaction is induced by cumulus cells, but not by the

ZP [Bedford et al. 2004]. The available evidence suggests a

general but not unique mechanism of penetration, and it is

important to consider particular species adaptations when

manipulating different samples in vitro. The sperm must

penetrate physical barriers imposed by the oocyte, including

cumulus oophorus cells, the plasma membrane, and the ZP,

for which hydrolytic enzymes such as glycohydrolases and

proteinases are necessary. During capacitation, the sperm

undergoes functional biochemical and biophysical modifica-

tions, including changes in the activity of membrane enzymes

and motility patterns, enabling it to undergo the AR prior to

fertilization. These modifications include the removal of

roadblocks to capacitation factors from the sperm surface and

increased membrane fluidity, cholesterol efflux, intracellular

Address correspondence to Jorge Parodi, Laboratorio de Fisiologıa de laReproduccion, Escuela de Medicina Veterinaria, Nucleo de Investigacionen Produccion Alimentaria, Facultad de Recursos Naturales, UniversidadCatolica de Temuco, Temuco, Chile. Tel: þ56-45-2205564. E-mail:jparodi@uct.cl

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calcium, cAMP, and protein tyrosine phosphorylation

[Aitken and McLaughlin 2007]. All of these processes

are regulated by the entry of calcium into the cells, but

another key factor is the motility of the sperm. The complete

process had previously been described as a single step:

the entry of calcium increases motility and the AR.

However, recently, the calcium wave concept has been

incorporated (see Figure 1; [Navarrete et al. 2010]),

indicating that in sperm cells, the first part of the calcium

wave generates an increase in motility, while the second

part induces the AR. Furthermore, this process can be

manipulated while the cells maintain a healthy state [Darszon

et al. 2011].

Capacitation factors

The reduction of factors inhibiting capacitation (disinhibit

factor, DF) due to the seminal flow involves a gradual

release of these factors, from the sperm surface. Their release

results in a transient state of sperm DF capacitation. This

ensures the maximum capacity of fertilization at the appro-

priate location [Acott and Carr 1984; Zhong et al. 1993].

Once the DF binds to the sperm surface, it activates a calcium

ATPase, thus maintaining a low calcium concentration.

When the DF is released from the sperm surface, an increase

in intracellular calcium levels is initiated. In vitro studies in

which the calcium ATPase was inhibited revealed acceler-

ation of capacitation [Perry et al. 1997].

Plasma membrane and ion channels

The plasma membrane is a lipoprotein interface that acts as a

permeability barrier allowing the cell to maintain a different

composition in the intracellular in comparison to the extra-

cellular medium. The most abundant components of the

plasma membrane are phospholipids and proteins, which

together form the fluid mosaic pattern [Hasdemir 2007].

The resting potential is a particular state of the membrane

potential in which the sum of ion currents through the

membrane is zero. This is due to the presence of transmem-

brane electrochemical gradients resulting from selective

permeability to ions and secondary various structures such

as transmembrane channels, pumps, and ion exchangers.

From the resting potential, cell excitation can generate an

action potential that allows the cell to respond to different

stimuli. During this process, each ion tends to draw the

membrane potential towards its own electrochemical equilib-

rium potential (Nernst equation) [Hille 1992]. Ionic currents

through channels determine transmembrane bioelectric

phenomena related to the membrane potential in addition

to modulating enzyme activity, metabolism, and cellular

genetics activity. Specifically, in sperm cells, the transmem-

brane ionic currents and their potential, among other factors,

regulate the intracellular concentration of calcium and the

genesis of second messengers. These factors are essential for

fertilization-associated processes, such as sperm motility,

capacitation, and the AR. Therefore, the study of ion channels

is extremely valuable for understanding the electrophysio-

logical processes and biological responses of both excitable

cells and isolated cells. In particular, determining the roles of

these channels in the mammalian sperm membrane is

essential to understand the processes involved in fertilization.

The main tool for investigating the characteristics and

distribution of ion channels in the plasma membrane is the

patch-clamp technique [Neher and Sakmann 1976; Neher

et al. 1978], which is a high-resolution method that is

currently used to determine the electrophysiological and

pharmacological properties of the cell structure.

Sperm cell viability and function

One must be careful during the various procedures in which

sperm are manipulated as alterations can cause premature

sperm capacitation [Gomez et al. 1997]. This leads to the

AR impacting the longevity of the sperm. A decrease in

fertilization capacity can result from the presence of large

amounts of ROS following ejaculation. Kirchhoff and asso-

ciates [1998] and Alvarez and Agarwal [2006] indicated

that sperm produce and export ROS to the extracellular

environment, most of which are generated by the mitochon-

dria, secondary to the flagellar activity of the cells. The loss

of sperm function, i.e., the fertilization capacity, results from

the presence of high levels of ROS, either following

ejaculation or secondarily to high levels of motility. These

studies have indicated that the sperm produced and exported

ROS to the extracellular environment are the product of the

monovalent reduction of molecular oxygen during oxidative

phosphorylation [Alvarez and Agarwal 2006; Kirchhoff et al.

1998]. Observations made in the laboratory in models

of immobile sperm cells (salmon or trout) have suggested

Figure 1. Calcium wave. The upper panel shows a bovine sperm with acalcium probe exposed to a high potassium concentration, while thelower panel shows a graphic representation of the fluorescence intensity,both in control conditions and when sperm are exposed to potassium.The figure indicates that there is a wave from the middle piece to thehead when the sperm are depolarized. Modified figure from Navarreteet al. 2010.

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the existence of long-term viability, lasting for days or weeks,

while retaining a high rate of fertilization. The common

feature of such models is the inactive state of the cells,

without metabolic changes, i.e., ROS production. In mam-

malian sperm cells, we have observed only a one-hour period

of viability and function, and these cells show a high motility

and metabolism. Temperature is important for the regulation

of cell function, and the preservation and the quality of sperm.

For example, fish semen display extreme temperature control

[Alavi and Cosson 2005], and in porcine sperm, temperature

conservation increases the time of preservation of a sample

[Althouse et al. 1998]. Furthermore, in fowl, temperature

regulates calcium influx [Thomson and Wishart 1991].

These lines of evidence suggest the importance of tempera-

ture control during the in vitro manipulation of sperm cells,

which is correlated with changes during travel in the

oviduct or in fresh water, in the case of the aquatic species.

In particular, there may be temperature gradients within the

oviduct of animals in estrous [Bahat and Eisenbach 2006;

Hunter and Nichol 1986]. Values presented in the literature

suggest that these gradients can be on the order of 1–2 �C or

more between the caudal portion of the isthmus and the

cranial portion of the ampulla in the hours before ovulation.

This has been proposed to contribute to reducing sperm

motility and the sperm storage function of the caudal isthmus

[Hunter and Nichol 1986]. The magnitude of the temperature

gradient may change according to the stage of the cycle and,

especially, according to the time of ovulation [Hunter 2012].

Therefore, there is a possible influence of temperature on the

viscosity and viscoelasticity of female tract fluids and on the

ZP, as in other cell models, membrane viscosity is affected

by temperature [Stokke et al. 1985]. This factor must be

considered, and it might be most significant at the time when

viable spermatozoa are expected to be found in the oviduct

[Coy et al. 2009]. Temperature is a key factor in the function

of sperm cells, and we can control it in vitro. Thus, we

observed natural changes in the oviduct when the sperm cells

are swimming towards the oocyte. Moreover, in aquatic

species, environmental conditions are vital to fecundity.

Kv currents identified in sperm

A previous study revealed the presence of different types

and differentially localized potassium channels [Darszon et al.

2006; Hagiwara and Kawa 1984]. An example is the delayed

rectifier Kþ type channel found in rat spermatogenic cells,

which shows a trend that is independent of extracellular

calcium and is blocked by tetraethylammonium chloride

(TEA) [Hagiwara and Kawa 1984]. Based on these charac-

teristics, we identified an inward rectifier Kþ channel referred

to as Kir [Munoz-Garay et al. 2001]. This channel is also

regulated by the intracellular pH, with an acidic intracellular

pH (6.3) inhibiting the current in spermatogenic cells, while a

rising intracellular pH (7.4) significantly increases conduct-

ance in these cells. We further identified a third type of Kþ

channel, designated mSlo3, which was cloned in rat

spermatogenic cells and has been expressed in Xenopus

laevis oocytes for biophysical analyses. Recent studies using

electrophysiological methods allowed an output current from

the sperm midpiece that is sensitive to TEA to be detected

[Marconi et al. 2008], and depolarization regulating calcium

entry was described.

Regulation of calcium voltage channels (CAVs)during capacitation

During capacitation, ionic channels are susceptible to being

activated when a change in the configuration of these

channels occurs and are mediated by a change in the

membrane potential. In rat and bovine sperm, the membrane

potential is between �10 and �50 mV [Clapham et al. 2003;

Darszon et al. 2005]. Low voltage calcium is inactivated at

these voltages and therefore does not respond to depolarizing

stimuli. Analysis of the membrane potential of rat sperma-

tozoa showed that only cells that maintain hyperpolarization

are able to generate an increased flow of calcium secondary

to contact with the ZP (likely secondary CAVs) and carry

out the RA [Arnoult et al. 1999]. Capacitation, resulting in

hyperpolarization, changes the configuration of the CAV in a

manner that is open to the agonist-mediated ion flow only at

a specific stage, thus avoiding early RA. Studies in sperm

conducted using electrophysiological methods have demon-

strated the role of calcium channel functional are keys in

capacitation, which are dependent on the membrane potential

[Darszon et al. 2005; Wennemuth et al. 2000]. However, the

complete mechanism underlying this phenomenon and its

regulation via calcium entry is not completely understood.

In this context, it was recently suggested that calcium entry

occurs via depolarization and the regulation of motility, with

a second entry event occurring due to pH regulation and

depolarization, and this second calcium influx is mediated by

the AR [Escoffier et al. 2007]. These findings have led to new

models in which not only the type of CatSper channel is

responsible for this phenomenon [Xia et al. 2007] but have

further allowed the electrophysiological investigation of new

phenomena, such as depolarization, that are also involved in

the regulation of these voltage-dependent calcium channels.

Cationic sperm (CatSper) channels

Four members of the CatSper channels have been described

(CatSper1-4) in murine sperm [Quill et al. 2001; Ren et al.

2001]. These channels consist of 6 transmembrane domains

(6TM1) that are voltage-dependent and calcium-permeable

and appear to be found only in sperm cells. CatSper1 and

2 channels have been reported to be essential for sperm

hyperactivation and fertility. However, reports concerning

these channels still mainly result from studies of humans and

mice [Clapham and Garbers 2005].

Functional features of the plasma membrane of the sperm

tail have been described [Ren et al. 2001]. Other reports have

localized these proteins to the principal piece of the flagellum

[Kirichok et al. 2006; Qi et al. 2007]. Additional evidence

regarding the distribution of CatSper in different species and

its localization in sperm cells is being obtained through

ongoing investigations, which is important for designing

solutions for the manipulation of samples. Studies in which

the expression of this protein has been manipulated have led

to the generation of a male sterile phenotype in a normal

mouse model. While the mating behavior, sperm counts, and

sperm cell morphology of these mutant mice are

DOI: 10.3109/19396368.2013.869273 Sperm cells viability 3

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indistinguishable from those of wild type mice, the CatSper1

mutant sperm cells are slow, exhibit a reduced basal rate, and

have no effect on the bathing or the bending of the tail region.

The mutant sperm cells cannot fertilize the eggs with an intact

zone pellucid but can fertilize eggs when the outer layers have

been enzymatically removed [Ren et al. 2001], suggesting

changes in some cell functions. Male mice lacking CatSper2

are also infertile due to a lack of the hyperactivated motility

required for penetration of the extracellular matrix of the

egg [Quill et al. 2003]. In a study in humans, subfertile men

with deficient sperm motility showed significantly reduced

expression of CatSper1 [Nikpoor et al. 2004]. Little is known

about CatSper3 and CatSper4, but they appear to be involved

in supporting cell functions in the sperm [Clapham and

Garbers 2005].

The above leads to two questions: (1) can these channels

explain all of the phenomena observed in the sperm cells?

(2) is there sufficient evidence to support the idea that

CatSper channels explain the entire model of the sperm

activity? It is accepted that CatSper channels and their various

isoforms are responsible for cellular functions in sperm.

Additionally, the relationship between CatSper and proges-

terone has been described, and the authors indicated the effect

of progesterone on increasing intracellular calcium levels

[Blackmore 1993; Turner and Meizel 1995]. While the

relationship between progesterone and CatSper has been

described [Lishko et al. 2011; Strunker et al. 2011], the

mechanism underlying the regulation of CatSper function

by progesterone is not completely understood, although the

intracellular PI3K-AKT signaling pathway was recently

implicated in this process. However, progesterone may be

associated with other receptors in sperm cells, such as

GABAa [Shi and Roldan 1995], or in the regulation of another

channel, such as potassium [Kumar et al. 2000], or voltage-

dependent calcium channels [Bonaccorsi et al. 2001].

Progesterone has been described to play a role in the specific

functions of sperm cell channels [Sagare-Patil et al. 2013].

Additionally, CatSper is modulated by pH [Fraire-Zamora and

Gonzalez-Martinez 2004] and bicarbonate [Wennemuth et al.

2003]. Nevertheless, additional events must be coordinated

for fecundation to occur successfully, including the AR, the

regulation of membrane stability, calcium signaling, and

mitochondrial function, among others, beyond Catsper modu-

lation. However, these events are not described in all models,

and other electrical phenomena can cooperate in the cellular

events described in sperm. A complete table of ion channels,

indicating the presence of voltage-dependent calcium chan-

nels and CatSper, in humans and mice is available [Darszon

et al. 2011]. This review indicates that we lack a complete

understanding of the localization of these channels, and there

are other mechanisms that may alter intracellular calcium.

Changes observed in the membrane potentialof sperm cells

An increase in the membrane potential, described as

hyperpolarization, occurs during capacitation in rat, bovine,

and human spermatozoa [Arnoult et al. 1996; Brewis et al.

2001; Zeng et al. 1996]. In rat sperm, hyperpolarization is the

result of increased permeability to Kþ [Zeng et al. 1995],

leading to a change in the membrane potential. During

capacitation, there is an increase in the pHi of more than

0.2 units [Zeng et al. 1996], which is sufficient to induce an

increase of 0.5 to 3 times in the probability of the opening

of Kir channels found in other tissues [Gutman et al. 2003].

Thus, under physiological conditions, an increase in pHi

activates Kir channels. It has been suggested that this process

hyperpolarizes the sperm membrane [Krasznai et al. 2000].

Furthermore, Kv-activated intracellular calcium is modulated

by the increase in the concentration of intracellular calcium

that occurs during capacitation, thus contributing to hyperpo-

larization [Jagannathan et al. 2002]. Together these observa-

tions confirm the role of Kþ currents in the hyperpolarization

of the sperm membrane and its effect on capacitation and the

subsequent AR. However, in other cell models, the mechan-

ism reflects blocking the Kþ channel shaft, depolarization,

and calcium channel opening [Baker et al. 1973; Wellman

et al. 2001]. In sperm models, it is accepted that Kir channels

are able to hyperpolarize the membrane, but these channels

are controlled by physiological phenomena, leading to changes

in the membrane potential and correcting this potential,

allowing positive charges to be relocated to restore balance

and maintain a physiological membrane potential [Gutman

et al. 2003]. Kv-type channels are present in sperm [Marconi

et al. 2008], and their current is modulated by peptides,

suggesting a means to modulate currents in sperm [Parodi

et al. 2010]. This model is sensitive to ASD and can be applied

to generate depolarization in other cell models, leading to an

increase in intracellular calcium levels and consequent cellular

changes [Navarrete et al. 2010]. Some evidence suggests that

this mechanism is part of a complex mechanism of regulation

that also includes the hyperpolarization and depolarization

described in sperm [Fraire-Zamora and Gonzalez-Martinez

2004; Gonzalez-Martinez 2003; Neri-Vidaurri Pdel et al.

2006], which can generate changes in the membrane potential,

causing an influx of calcium and alterations in the physiology

of sperm [Babcock and Pfeiffer 1987; Linares-Hernandez et al.

1998]. It is not hyperpolarization alone that mediates this

phenomenon. The control of the membrane potential of sperm

cells can block calcium entry and the associated secondary

signaling. Many drugs can block changes in the membrane

potential; could these drugs be used as potential regulators of

sperm motility? A high concentration of potassium can induce

changes in intracellular calcium levels, in the form of a wave

from the middle piece to the head of the sperm. Figure 1 shows

the effect of high potassium on intracellular calcium levels in

bovine sperm (from [Navarrete et al. 2010]).

Calcium as a second messenger

The processes that generate second messengers that regulate

cellular physiology have been studied for several years.

Calcium is important for the regulation of kinase activity,

phosphatases, gene activation, and protein translation. It is

required at high concentrations for short periods of time,

and cells display various mechanisms for finely regulating its

intracellular concentration and maintaining a physiological

calcium gradient [Hurwitz 1996; Stewart 1985]. Thus, various

signals transiently increase intracellular calcium, which is

indicative of activation of cellular processes, whereas a

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sustained increase in the cellular concentration indicates cell

toxicity [Pounds 1984]. These changes in intracellular

calcium concentrations may vary depending on cell type

and the type of stimulation involved. Furthermore, they have

been correlated with the process of vesicular exocytosis

based on observations made through different techniques for

measuring calcium currents and cell capacitance [Trifaro

et al. 2000], including fluorometric measurements of calcium

levels and amperometric records [Elhamdani et al. 1994].

For example, the normal process of vesicle release is

highly dependent on calcium entry, which is crucial for the

propagation of nerve impulses and the establishment of neural

connections responsible for cognitive brain functions. In

sperm cells, the intracellular activation of vesicles in the

AR is similar to what is observed in other types of somatic

cells and depends on changes in calcium levels. These

findings suggest that different pathways leading to changes

in calcium levels play a role in the development of different

models of cell physiology.

Animal species of industrial interest

Understanding the influence of reproduction in food produc-

tion is important in relation to increasing output and yield

as well as maintaining and preserving genetic markers to

improve productivity. Regarding the production of meat for

consumption, cows, goats, pigs, and fish have been instru-

mental in the development of this industry. In recent years,

assisted reproduction has begun to be applied in these species

by preserving oocytes and sperm for later use in artificial

insemination. The main reference models studied have been

mice and humans, and similar techniques have been imple-

mented in cows. Work aimed at the cryopreservation of sperm

from salmon and other species was recently initiated, with

sperm being frozen for transport, storage, and handling. There

is high national and international demand for animal repro-

duction, as the meat market is steadily increasing, and the

requirements for animal protein for human populations are

also increasing [Food and Agriculture Organization of the

United Nations, 2003]. The world population in 2030 will

consume more and better food, with 3050 kilocalories (kcal)

being available per person, compared to 2360 kcal per person/

day in the mid-1960s and the 2800 kcal available currently.

This change reflects the increase in consumption in many

developing countries, whose average daily intake will be

approximately 3000 kcal in 2030. For example, it has been

reported that the domestic consumption of pork per person

has increased [Oficina de Estudios y Politicas Agrarias,

2011], reaching values of 23 kg/capita in recent years. Thus,

pork has become the second most commonly consumed meat,

while poultry consumption decreased from 2000 to 2006 and

has remained even at levels of 18 kg/capita over the last 4

years. The economic returns from the exploitation of animal

flesh under current market conditions are based on the

management of their genes and the use of high-genetic value

players together with the best production techniques to obtain

high-quality meat products at competitive cost. Reproduction

is one of the most important aspects of the animal resource, as

it allows the continuity of the species to be maintained.

Additionally, the economic importance of reproductive

behavior in cattle is well-known. Ingvartsen and Moyes

[2013] summarized that essential studies examining the

factors that affect the same traits will increase productivity

in females. Thus, techniques including the control of insem-

ination have begun to be viewed as an alternative for

improving production, and the discussion regarding pheno-

typic traits of importance to the industry is increasing.

How do we maintain these gametes, increase cell function,

and apply these techniques under various industrial condi-

tions? This is not an easy question to answer, but the cellular

functions of sperm related to generating such compounds as

well as protocols and conditions applicable in this industry

should be determined.

Mature sperm cells are complex cellular machines that

through a series of steps and environments reach their target,

the oocyte, and fulfill the purpose of delivering their genetic

material via fertilization. In this review, we have highlighted

flagellar motility and capacitation, which is characterized

by the AR. In recent years, the function of CatSper channels

as regulatory elements has shown to be indirectly involved in

modulating the motility and fertilization capacity of sperm as

well as calcium entry. A recent study has now demonstrated

that a CatSper channel is involved in the motility but not in

the AR [Sagare-Patil et al. 2013]. Flagellar movement

generates various changes, including the production of ROS,

and these increases can explain the reduction of cell viability.

Moreover, some sperm cell models can remain immobile for a

period of time. These sperm cells show a long period of

viability and maintain their cellular functions for days. When

activated, the cells become motile upon external signaling

(i.e., osmotic changes). Calcium regulation is important for

the general function of cells. In mammalian sperm cells, a

recent study has suggested that there are two steps regulated

by calcium entry: first, the motility of sperm cells, and

second, the AR. Since motility generates ROS it is

hypothesized here that regulation by calcium reduces the

motility and the general metabolic state of the cells, leading to

a reduction of cell mortality. All of these regulatory

mechanisms are important for the conservation and manipu-

lation of sperm cells. Because food production, and especially

that of animal protein, has increased in recent decades,

reproductive processes must be understood to provide an

efficient means of control. It is vital for the development of

the food industry to study these processes, yet little is known

about the cells involved and the conditions that must occur.

Thus, we should study other species as a reference for the

development and maintenance of sperm as a function of

process.

Declaration of interest

The author reports no conflicts of interest. The author alone

is responsible for the content and writing of the paper.

References

Acott, T.S., and Carr, D.W. (1984) Inhibition of bovine spermatozoa bycaudal epididymal fluid: II. Interaction of pH and a quiescence factor.Biol Reprod 30:926–35.

Aitken, R.J., and McLaughlin, E.A. (2007) Molecular mechanismsof sperm capacitation: progesterone-induced secondary calcium

DOI: 10.3109/19396368.2013.869273 Sperm cells viability 5

Syst

Bio

l Rep

rod

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from

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care

.com

by

201.

186.

171.

150

on 1

2/13

/13

For

pers

onal

use

onl

y.

oscillations reflect the attainment of a capacitated state. Soc ReprodFertil Suppl 63:273–93.

Alavi, S.M., and Cosson, J. (2005) Sperm motility in fishes. I. Effectsof temperature and pH: a review. Cell Biol Int 29:101–10.

Althouse, G.C., Wilson, M.E., Kuster, C., and Parsley, M. (1998)Characterization of lower temperature storage limitations of fresh-extended porcine semen. Theriogenology 50:535–43.

Alvarez, J.G., and Agarwal, A. (2006) Development of a novel homesperm test - what are the limitations? Hum Reprod 21:3029–30;author reply 3030–3031.

Arnoult, C., Kazam, I.G., Visconti, P.E., Kopf, G.S., Villaz, M.,and Florman, H.M. (1999) Control of the low voltage-activatedcalcium channel of mouse sperm by egg ZP3 and by membranehyperpolarization during capacitation. Proc Natl Acad Sci USA 96:6757–62.

Arnoult, C., Zeng, Y., and Florman, H.M. (1996) ZP3-dependentactivation of sperm cation channels regulates acrosomal secretionduring mammalian fertilization. J Cell Biol 134:637–45.

Babcock, D.F., and Pfeiffer, D.R. (1987) Independent elevation ofcytosolic [Ca2þ] and pH of mammalian sperm by voltage-dependentand pH-sensitive mechanisms. J Biol Chem 262:15041–7.

Bahat, A., and Eisenbach, M. (2006) Sperm thermotaxis. Mol CellEndocrinol 252:115–19.

Baibakov, B., Gauthier, L., Talbot, P., Rankin, T.L., and Dean, J. (2007)Sperm binding to the zona pellucida is not sufficient to induceacrosome exocytosis. Development 134:933–43.

Baker, P.F., Meves, H., and Ridgway, E.B. (1973) Calcium entry inresponse to maintained depolarization of squid axons. J Physiol 231:527–48.

Bedford, J.M., Mock, O.B., and Goodman, S.M. (2004) Novelties ofconception in insectivorous mammals (Lipotyphla), particularlyshrews. Biol Rev Camb Philos Soc 79:891–909.

Blackmore, P.F. (1993) Rapid non-genomic actions of progesteronestimulate Ca2þ influx and the acrosome reaction in human sperm.Cell Signal 5:531–8.

Bonaccorsi, L., Forti, G., and Baldi, E. (2001) Low-voltage-activated calcium channels are not involved in capacitation andbiological response to progesterone in human sperm. Int J Androl 24:341–51.

Breitbart, H. (2003) Signaling pathways in sperm capacitation andacrosome reaction. Cell Mol Biol (Noisy-le-grand) 49:321–7.

Brewis, I.A., Morton, I.E., Moore, H.D., and England, G.C. (2001)Solubilized zona pellucida proteins and progesterone induce calciuminflux and the acrosome reaction in capacitated dog spermatozoa.Mol Reprod Dev 60:491–7.

Cabrita, E., Anel, L., and Herraez, M.P. (2001) Effect of externalcryoprotectants as membrane stabilizers on cryopreserved rainbowtrout sperm. Theriogenology 56:623–35.

Clapham, D.E., and Garbers, D.L. (2005) International Union ofPharmacology. L. Nomenclature and structure-function relationshipsof CatSper and two-pore channels. Pharmacol Rev 57:451–4.

Clapham, D.E., Montell, C., Schultz, G., and Julius, D. (2003)International Union of Pharmacology. XLIII. Compendium ofvoltage-gated ion channels: transient receptor potential channels.Pharmacol Rev 55:591–6.

Coy, P., Gadea, J., Rath, D., and Hunter, R.H. (2009) Differing spermability to penetrate the oocyte in vivo and in vitro as revealed usingcolloidal preparations. Theriogenology 72:1171–9.

Darszon, A., Acevedo, J.J., Galindo, B.E., Hernandez-Gonzalez, E.O.,Nishigaki, T., Trevino, C.L., et al. (2006) Sperm channel diversityand functional multiplicity. Reproduction 131:977–88.

Darszon, A., Nishigaki, T., Beltran, C., and Trevino, C.L. (2011)Calcium channels in the development, maturation, and function ofspermatozoa. Physiol Rev 91:1305–55.

Darszon, A., Nishigaki, T., Wood, C., Trevino, C.L., Felix, R., andBeltran, C. (2005) Calcium channels and Ca2þ fluctuations in spermphysiology. Int Rev Cytol 243:79–172.

Elhamdani, A., Bossu, J.L., and Feltz, A. (1994) Evolution of the Ca2þcurrent during dialysis of isolated bovine chromaffin cells: effect ofinternal calcium. Cell Calcium 16:357–66.

Escoffier, J., Boisseau, S., Serres, C., Chen, C.C., Kim, D., Stamboulian,S., et al. (2007) Expression, localization and functions in acrosomereaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels insperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficientmice. J Cell Physiol 212:753–63.

Food and Agriculture Organization of the United Nations. (2003) Proteinand amino acid requirements in human nutrition: report of a jointFAO/WHO/UNU expert consultation. Author; Geneva.

Fraire-Zamora, J.J., and Gonzalez-Martinez, M.T. (2004) Effect ofintracellular pH on depolarization-evoked calcium influx in humansperm. Am J Physiol Cell Physiol 287:C1688–96.

Gomez, M.C., Catt, J.W., Gillan, L., Evans, G., and Maxwell, W.M.(1997) Effect of culture, incubation and acrosome reaction of freshand frozen-thawed ram spermatozoa for in vitro fertilization andintracytoplasmic sperm injection. Reprod Fertil Dev 9:665–73.

Gonzalez-Martinez, M.T. (2003) Induction of a sodium-dependentdepolarization by external calcium removal in human sperm. J BiolChem 278:36304–10.

Gutman, G.A., Chandy, K.G., Adelman, J.P., Aiyar, J., Bayliss, D.A.,Clapham, D.E., et al. (2003) International Union of Pharmacology.XLI. Compendium of voltage-gated ion channels: potassium channels.Pharmacol Rev 55:583–6.

Hagiwara, S., and Kawa, K. (1984) Calcium and potassium currentsin spermatogenic cells dissociated from rat seminiferous tubules.J Physiol 356:135–49.

Hasdemir, U. (2007) The role of cell wall organization and active effluxpump systems in multidrug resistance of bacteria. Mikrobiyol Bul 41:309–27.

Hille, B. (1992) Ionic channels of excitable membranes. Sinauer;Sunderland, MA, Chapter 3, 68p.

Hunter, R.H. (2012) Temperature gradients in female reproductivetissues. Reprod Biomed Online 24:377–80.

Hunter, R.H., and Nichol, R. (1986) A preovulatory temperature gradientbetween the isthmus and ampulla of pig oviducts during the phaseof sperm storage. J Reprod Fertil 77:599–606.

Hurwitz, S. (1996) Homeostatic control of plasma calcium concentra-tion. Crit Rev Biochem Mol Biol 31:41–100.

Ingvartsen, K.L. and Moyes, K. (2013) Nutrition, immune function andhealth of dairy cattle. Animal 7(Suppl 1):112–122.

Jagannathan, S., Publicover, S.J., and Barratt, C.L. (2002) Voltage-operated calcium channels in male germ cells. Reproduction 123:203–15.

Kirchhoff, C., Osterhoff, C., Pera, I., and Schroter, S. (1998) Function ofhuman epididymal proteins in sperm maturation. Andrologia 30:225–32.

Kirichok, Y., Navarro, B., and Clapham, D.E. (2006) Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activatedCa2þ channel. Nature 439:737–40.

Knobil, E. and Neill, J.D. (1994) The Physiology of reproduction. RavenPress; New York, 2nd ed, Chapter 1.

Krasznai, Z., Marian, T., Izumi, H., Damjanovich, S., Balkay, L.,Tron, L., et al. (2000) Membrane hyperpolarization removes inacti-vation of Ca2þ channels, leading to Ca2þ influx and subsequentinitiation of sperm motility in the common carp. Proc Natl Acad SciUSA 97:2052–7.

Kumar, S., Ying, Y.K., Hong, P., and Maddaiah, V.T. (2000) Potassiumincreases intracellular calcium simulating progesterone action inhuman sperm. Arch Androl 44:93–101.

Linares-Hernandez, L., Guzman-Grenfell, A.M., Hicks-Gomez, J.J., andGonzalez-Martinez, M.T. (1998) Voltage-dependent calcium influxin human sperm assessed by simultaneous optical detection ofintracellular calcium and membrane potential. Biochim BiophysActa 1372:1–12.

Lishko, P.V., Botchkina, I.L., and Kirichok, Y. (2011) Progesterone acti-vates the principal Ca2þ channel of human sperm. Nature 471:387–91.

Marconi, M., Sanchez, R., Ulrich, H., and Romero, F. (2008) Potassiumcurrent in mature bovine spermatozoa. Syst Biol Reprod Med 54:231–9.

Martinez-Lopez, P., Santi, C.M., Trevino, C.L., Ocampo-Gutierrez, A.Y.,Acevedo, J.J., Alisio, A., et al. (2009) Mouse sperm K+ currentsstimulated by pH and cAMP possibly coded by Slo3 channels.Biochem Biophys Res Commun 381:204–209.

Munoz-Garay, C., De la Vega-Beltran, J.L., Delgado, R., Labarca, P.,Felix, R., and Darszon, A. (2001) Inwardly rectifying K(þ) channelsin spermatogenic cells: functional expression and implication in spermcapacitation. Dev Biol 234:261–74.

Navarrete, P., Martinez-Torres, A., Gutierrez, R.S., Mejia, F.R., andParodi, J. (2010) Venom of the Chilean Latrodectus mactansalters bovine spermatozoa calcium and function by blocking theTEA-sensitive K(þ) current. Syst Biol Reprod Med 56:303–10.

6 J. Parodi Syst Biol Reprod Med, Early Online: 1–7

Syst

Bio

l Rep

rod

Med

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

201.

186.

171.

150

on 1

2/13

/13

For

pers

onal

use

onl

y.

Neher, E., and Sakmann, B. (1976) Noise analysis of drug inducedvoltage clamp currents in denervated frog muscle fibres. J Physiol258:705–29.

Neher, E., Sakmann, B., and Steinbach, J.H. (1978) The extracellularpatch clamp: a method for resolving currents through individual openchannels in biological membranes. Pflugers Arch 375:219–28.

Neri-Vidaurri Pdel, C., Torres-Flores, V., and Gonzalez-Martinez, M.T.(2006) A remarkable increase in the pHi sensitivity of volt-age-dependent calcium channels occurs in human sperm incubatedin capacitating conditions. Biochem Biophys Res Commun 343:105–9.

Nikpoor, P., Mowla, S.J., Movahedin, M., Ziaee, S.A., and Tiraihi, T.(2004) CatSper gene expression in postnatal development of mousetestis and in subfertile men with deficient sperm motility. Hum Reprod19:124–8.

Oficina de Estudios y Politicas Agrarias. (2011) Existencia de cerdosen criaderos por tipo, segun semestre. Available at: http://www.odepa.cl/articulos/MostrarDetalle.action;jsessionid=0144A1D0090E0066E0DB26825978CE0C?idcla=12&idn=4120 [last accessed 6 Dec 2013].

Parodi, J., Navarrete, P., Marconi, M., Gutierrez, R.S., Martinez-Torres,A., and Mejias, F.R. (2010) Tetraethylammonium-sensitive K(þ)current in the bovine spermatozoa and its blocking by the venom ofthe Chilean Latrodectus mactans. Syst Biol Reprod Med 56:37–43.

Patrat, C., Auer, J., Fauque, P., Leandri, R.L., Jouannet, P., and Serres, C.(2006) Zona pellucida from fertilised human oocytes induces avoltage-dependent calcium influx and the acrosome reaction inspermatozoa, but cannot be penetrated by sperm. BMC Dev Biol 6:59.

Perry, R.L., Barratt, C.L., Warren, M.A., and Cooke, I.D. (1997)Response of human spermatozoa to an internal calcium ATPaseinhibitor, 2,5-di(tert-butyl) hydroquinone. J Exp Zool 279:284–90.

Pounds, J.G. (1984) Effect of lead intoxication on calcium homeostasisand calcium-mediated cell function: a review. Neurotoxicology 5:295–331.

Qi, H., Moran, M.M., Navarro, B., Chong, J.A., Krapivinsky, G.,Krapivinsky, L., et al. (2007) All four CatSper ion channel proteins arerequired for male fertility and sperm cell hyperactivated motility.Proc Natl Acad Sci USA 104:1219–23.

Quill, T.A., Ren, D., Clapham, D.E., and Garbers, D.L. (2001) A voltage-gated ion channel expressed specifically in spermatozoa. Proc NatlAcad Sci USA 98:12527–31.

Quill, T.A., Sugden, S.A., Rossi, K.L., Doolittle, L.K., Hammer, R.E.,and Garbers, D.L. (2003) Hyperactivated sperm motility driven byCatSper2 is required for fertilization. Proc Natl Acad Sci USA 100:14869–74.

Ren, D., Navarro, B., Perez, G., Jackson, A.C., Hsu, S., Shi, Q., et al.(2001) A sperm ion channel required for sperm motility and malefertility. Nature 413:603–9.

Rossato, M., Di Virgilio, F., Rizzuto, R., Galeazzi, C., and Foresta, C.(2001) Intracellular calcium store depletion and acrosome reaction inhuman spermatozoa: role of calcium and plasma membrane potential.Mol Hum Reprod 7:119–28.

Sagare-Patil, V., Vernekar, M., Galvankar, M., and Modi, D. (2013)Progesterone utilizes the PI3K-AKT pathway in human spermatozoa

to regulate motility and hyperactivation but not acrosome reaction.Mol Cell Endocrinol 374:82–91.

Shi, Q.X., and Roldan, E.R. (1995) Evidence that a GABAA-likereceptor is involved in progesterone-induced acrosomal exocytosis inmouse spermatozoa. Biol Reprod 52:373–81.

Stewart, A.F. (1985) Calcium metabolism without anguish.Understanding the body’s homeostatic ‘black box’. Postgrad Med77:283–91, 294.

Stokke, B.T., Mikkelsen, A., and Elgsaeter, A. (1985) Human erythro-cyte spectrin dimer intrinsic viscosity: temperature dependence andimplications for the molecular basis of the erythrocyte membrane freeenergy. Biochim Biophys Acta 816:102–10.

Strunker, T., Goodwin, N., Brenker, C., Kashikar, N.D., Weyand, I.,Seifert, R., et al. (2011) The CatSper channel mediates progesterone-induced Ca2þ influx in human sperm. Nature 471:382–6.

Thomson, M.F., and Wishart, G.J. (1991) Temperature-mediatedregulation of calcium flux and motility in fowl spermatozoa.J Reprod Fertil 93:385–91.

Trifaro, J., Rose, S.D., Lejen, T., and Elzagallaai, A. (2000) Twopathways control chromaffin cell cortical F-actin dynamics duringexocytosis. Biochimie 82:339–52.

Turner, K.O., and Meizel, S. (1995) Progesterone-mediated efflux ofcytosolic chloride during the human sperm acrosome reaction.Biochem Biophys Res Commun 213:774–80.

Wellman, G.C., Cartin, L., Eckman, D.M., Stevenson, A.S.,Saundry, C.M., Lederer, W.J., et al. (2001) Membrane depolarization,elevated Ca(2þ) entry, and gene expression in cerebralarteries of hypertensive rats. Am J Physiol Heart Circ Physiol 281:H2559–67.

Wennemuth, G., Carlson, A.E., Harper, A.J., and Babcock, D.F. (2003)Bicarbonate actions on flagellar and Ca2þ -channel responses: initialevents in sperm activation. Development 130:1317–26.

Wennemuth, G., Westenbroek, R.E., Xu, T., Hille, B., and Babcock, D.F.(2000) CaV2.2 and CaV2.3 (N- and R-type) Ca2þ channels indepolarization-evoked entry of Ca2þ into mouse sperm. J Biol Chem275:21210–7.

Xia, J., Reigada, D., Mitchell, C.H., and Ren, D. (2007) CATSPERchannel-mediated Ca2þ entry into mouse sperm triggers a tail-to-head propagation. Biol Reprod 77:551–9.

Zeng, Y., Clark, E.N., and Florman, H.M. (1995) Sperm mem-brane potential: hyperpolarization during capacitation regu-lates zona pellucida-dependent acrosomal secretion. Dev Biol 171:554–63.

Yanagimachi, R. (2011) Mammalian sperm acrosome reaction:where does it begin before fertilization? Biol Reprod 85:4–5.

Zeng, Y., Oberdorf, J.A., and Florman, H.M. (1996) pH regulationin mouse sperm: identification of Na(þ)-, Cl(�)-, and HCO3(�)-dependent and arylaminobenzoate-dependent regulatory mechanismsand characterization of their roles in sperm capacitation. Dev Biol173:510–20.

Zhong, C.L., Xin, X.H., and Shi, Q.X. (1993) Inhibition of spermine oncalcium influx during capacitation of guinea pig spermatozoa in vitro.Zhongguo Yao Li Xue Bao 14:141–4.

DOI: 10.3109/19396368.2013.869273 Sperm cells viability 7

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