Reprod Dom Anim 44, 345349 (2009); doi: 10.1111/j.1439-0531.2008.01211.

x
ISSN 0936-6768

Review Article
Mitochondria in Mammalian Sperm Physiology and Pathology:
A Review
FJ Pena1, H Rodr guez Mart nez2, JA Tapia1, C Ortega Ferrusola1, L Gonzalez Fernandez1 and B Mac as Garc a1
1
Laboratory of Spermatology, Veterinary Teaching Hospital, University of Extremadura, Caceres, Spain; 2Division of Reproduction, Faculty of
Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden

Contents
While, for a long time, the role of mitochondria in sperm
physiology and pathology has been largely ignored, recent
research points out the mitochondria as a major organelle with
key roles in sperm function both under physiological and
biotechnological conditions. This paper briey reviews these
novel ndings regarding the role of mitochondria in sperm,
paying special attention to the most practical, readily applicable, aspects of the topic such as their role as a major source
of the sublethal damage that sperm experiments after cryopreservation.

Introduction
The spermatozoon is a highly specialized cell, whose main
function is to transport the male haploid genome in the
female genital tract and to deliver it during fertilization of
the oocyte. During their generation in the testis, the
spermatozoa loose most of the cell organelles, exception
of the acrosome (derived from the Golgi vesicle) and a
certain sub-set of mitochondria, which are allocated in
the mid-piece of the sperm tail in the mature spermatozoon, the latter playing critical roles in sperm function
prior to fertilization itself. The aim of the present review is
to give an overview of recent research on sperm
mitochondria, pointing out its function as regulators of
important sperm functions and their role in the origin
of the sub-lethal damage that the surviving population of
cryopreserved sperm experiments post-thaw.

Role of Mitochondria in Sperm Physiology

Mitochondria and sperm motility
Spermatozoa are able to generate energy (as adenosintriphosphate, ATP) through either aerobic or anaerobic metabolic pathways. Once monosaccharides,
often the major substrate form available for spermatozoa both in vivo or during in vitro handling, have
been transformed to glucose 6 phosphate (G 6-P), the
molecule enters the glycolitic pathway to generate
pyruvate. Later, pyruvate can either produce extracellular lactate or enter the mitochondrial Krebs cycle.
The control of this crossroad direction is elicited by
the enzyme lactate dehydrogenase (LDH), obeying the
concentration of substrates and products. Metabolization of lactate through the Krebs cycle yields much
more amounts of ATP, than glycolisis, and also
produces high amounts of reducing potential in the
form of nicotinamide adenine dinucleotide hydro-

genase (NADPH). The latter is of great importance

in the maintenance of anabolic pathways and in the
control of intracellular redox potential and pH. The
equilibrium between glycolisis and glycolisis-oxidative
phosphorylation depends on factors such as the O2
pressure, intracellular levels of ATP and a number of
intracellular factors such as nitric oxide. Traditionally,
it has been considered that the main role of the sperm
mitochondria is the production of energy for sperm
motility through oxidative phosphorylation. Currently,
this concept is under revision and debate (Marin et al.
2003; Miki et al. 2004; Mukai and Okuno 2004;
Rodr guez Gil 2006).
Mitochondria are found only in the mid-piece, thus
oxidative phosphorylation occurs only at this level.
However, agelar kinases and dynein-ATPases need
large amounts of ATP to maintain sperm motility all
along the agellum (Cao et al. 2006). It is has, therefore,
been suggested that the amount of ATP produced in the
mitochondria is not enough to diuse all along the
agellum to provide enough energy to support motility
(Turner 2003). In addition, studies in mice have demonstrated that defective oxidative phosphorylation does
not inhibit sperm motility (Escalier 2006). Recent
research demonstrates that boar spermatozoa incorporate a very small amount of the produced lactate into the
Krebs cycle (Marin et al. 2003). As well, glycolitic
enzymes have been identied in the principal piece
agellum in mammalian spermatozoa, including hexokinase, LDH and glyceraldehydes 3- phosphate dehydrogenase (GADP-S) (Nagdas et al. 2005, 2006; Perl
et al. 2006). Discrepancies exist in the current literature
regarding the importance of glycolisis or Krebs cycle as
energy sources for sperm motility; apparently, there are
species-specic dierences, with boars having the highest glycolitic activity (Marin et al. 2003). Spermatozoa
from bulls, but not those of mice, depend on the Krebs
cycle to maintain sperm motility (Aitken et al. 2004;
Mukai and Okuno 2004). However, all these studies
have focused on activated motility, and leave hyperactivated motility, another kinematic characteristic of the
spermatozoon under an eventual dierent regulatory
process (Rodr guez Gil 2006). Noteworthy, the immense
majority of the studies regarding motility changes have
been performed in vitro, and what we see in vitro
might not reect the physiological conditions that the
spermatozoa experience within the female reproductive
tract.

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FJ Pena, H Rodr guez Mart nez, JA Tapia, C Ortega Ferrusola, L Gonzalez Fernandez and B Mac as Garc a

Mitochondria and sperm viability: regulation of cell death

The second major function of the sperm mitochondria is
the regulation of cell death (Erkkila et al. 2006; Lesnefsky and Hoppel 2006; Ott et al. 2007), by being the
major source of reactive oxygen species (ROS), mainly
generated at complexes I and III of the respiratory chain
(Krahenbuhl et al. 1991; Gonzalvez and Gottlieb 2007).
The concentration of superoxide anion in the mitochondria is about 5- to 10-fold higher than in the cytosol
or nucleus (Cadenas and Davies 2000), and thus
mitochondria might also be a primary target of the
damage generated by ROS. Mitochondria-generated
ROS play an important role in the release of
cytocrome-c and other pro-apoptotic proteins, which
can trigger caspase activation and apoptosis (Mishra
and Shana 2005; Chan 2006; Tsujimoto and Shimizu
2007). Apoptosis is a complex phenomenon regulating
cellular proliferation. The process can be divided into
three phases: induction, execution and degradation.
After induction of apoptosis, mitochondrial pores are
opened, leading to a decreased mitochondrial membrane
potential (DYm). Opening of mitochondrial pores causes
the release of pro-apoptotic factors in the cytoplasm
where they are activated leading to the degradation
phase. During this phase (the degradation phase),
changes such as an increase in the permeability of the
sperm plasma membrane, and the externalization of the
constituent phospholipid phosphatidylserine (which
triggers a non-inammatory recognition of the apoptotic cells by phagocyte cells) occur.
When the permeability of the inner mitochondrial
membrane increases for solutes smaller than 1.5 kD, it is
assumed that the permeability transition pore is opened
(Grimm and Brdiczka 2007). When this occurs, the
mitochondrial membrane potential (DYm), which relies in
the impermeability of the inner mitochondrial membrane
for protons, breaks down, and with it, the ability of the
cell to synthesize ATP. The ensuing blockade of the
respiratory chain leads to the generation of ROS intermediates, most likely via direct transfer of electrons to
molecular oxygen (Grimm and Brdiczka 2007). In addition, the high concentration of solutes in the mitochondrial matrix generates an osmotic pressure, which drives
the inux of water molecules and the expansion of the
inner mitochondrial membrane, eventually disrupting the
outer membrane leading to the release of pro-apoptotic
factors that nally ends in the demise of the cell as a whole.
Mitochondria play also a crucial role in diverse cellular
functions such as energy production, modulation of redox
status, osmotic regulation and Ca++ homeostasis.

Mitochondria and epididymal sperm maturation and

fertilization
Recent research indicates that specic changes in the
mitochondria are critical steps of sperm maturation in
the epididymis (Aitken et al. 2004). Tyrosine phosphorylation, rst in the mid-piece and later in the tail, is a
major step in sperm maturation in the epididymis. This
change is associated with the phosphorylation of
several mitochondrial proteins, creation of mitochondrial membrane potential and activation of mito-

chondrial free-radical production (Aitken and Baker

2006; Aitken et al. 2007). In sea urchin, sperm fertilization is associated with mitochondrial deformation and
phosphatidylserine exposure (Kazama et al. 2006). On
the contrary, studies in humans demonstrate that
maintenance of high mitochondrial membrane potential
is necessary for a sperm to be fertile (Gallon et al. 2006).
Until recently, it was widely accepted that spermatozoa were translationally silent, an axiom that was
questioned by the demonstration of 55 s mitochondrial
ribosomes being actively involved in protein translation
in human, rat, mouse and bovine spermatozoa (Gur and
Breitbart 2006). Moreover, inhibition of protein translation signicantly reduced sperm motility, capacitation
and in vitro fertilization rate in rats, mice and humans
(Miller and Ostermeier 2006; Miller 2007).

Role of Mitochondria in Sperm Pathology

Mitochondria and abnormal spermatozoa
In a number of infertility or sub-fertility cases, both
under experimental and clinical situations, mitochondria
play a major role. Selenium deciency in the murine
species is associated with mitochondrial swelling and
loss of contact of the mitochondrial helix with the
plasma membrane and losses of mitochondria (Shalini
and Bansal 2007). In asthenozoospermic humans, the
respiratory eciency is reduced (Ferramosa et al. 2007).
In addition, morphological alterations of the mitochondria have been described in asthenozooespermic
patients (Rawe et al. 2007).
Role of mitochondria in the damage induced by
cryopreservation (the theory of premature aging of
cryopreserved spermatozoa)
After thawing, even using adequate cryopreservation
protocols, on an average only 50% of the entering sperm
population survive the process, when diagnosing this
survival as the percentage of spermatozoa maintaining
motility and membrane integrity (Rodr guez Mart nez
2007). Theoretically, the number of live spermatozoa
present in a FT-sample should be enough to give good
pregnancy rates, specially bearing in mind that FTsamples usually contain large numbers of spermatozoa
to compensate the well-known cell death that occurs
during cryopreservation. However, in most instances,
this is not the case. Hence, the question is: Why do FTspermatozoa have lower fertility, although they can
maintain good motility and membrane integrity rates
after cryopreservation? To answer this question, we
have to focus on the surviving population of spermatozoa. Cryopreservation may result in a population of
partly damaged, but yet motile spermatozoa. Such subpopulation often exhibits a certain degree of plasma and
mitochondrial leakiness, which may be accompanied by
lower energy metabolites and excessive energy losses
(Oehninger et al. 2000; Pena et al. 2003, 2005, 2006;
Martin et al. 2004, 2007; Mart nez-Pastor et al. 2004).
Those spermatozoa may very well become uncapacitated and loss their fertilizing ability within a few hours.
This fact is critical in species such as pigs and horses,
having long oestrous periods. If the time interval from

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Mitochondria in Mammalian Sperm Physiology and Pathology

articial insemination (AI) to ovulation exceeds the

lifespan of the cryopreserved spermatozoa, fertilization
will hardly occur. In species such as cows, having short
oestrus periods, AI with frozen-thawed semen is widely
and successfully used mainly because this short lifespan
of cryopreserved spermatozoa can be easily compensated by a AI close to ovulation, without the need of
time-consuming or expensive procedures. Simply, a
good oestrus management allows performing AIs close
enough to ovulation. However, in other species, this is
not the case unless oestrus control performed by
capacitated personnel, which is not always feasible,
particularly when considering large piggeries and the
economic constrains the sector has. Good pregnancy
rates have been obtained in pigs inseminated with FTspermatozoa when, for instance, frequent ultrasound
examinations are performed and AI is performed within
)4 to 0 h of the ovulation (Wongtawan et al. 2006).
However, this technique is impractical and extremely
expensive to be used under commercial farm conditions.
For years, it has been assumed that this short lifespan
of spermatozoa following cooling and freezing-thawing
was due to a premature capacitation (Watson 2000). In
following years, the scientic community found that the
process albeit resembling capacitation to some extent
diered from the real capacitation (Pena et al. 2004;
Bravo et al. 2005) and therefore it has to be considered a
dierent phenomenon when looking at the capacitation-like changes occurring during cryopreservation.
Some of these are the increase in intracellular Ca++
concentration in the spermatozoa (Watson 2000). Probably the extensive use of the CTC staining in the past,
whose basis is not yet fully understood, but that check
changes in intracellular Ca++ (Bravo et al. 2005), has
lead to the currently considered erroneous theory of
cryocapacitation. However, it is clear that cryopreserved
spermatozoa have a reduced lifespan both in vitro and
in vivo, a fact that largely constrain the use of FT-semen,
especially in animals showing long oestrus periods.
As stated earlier, mitochondria are cellular organelles
regulating cell survival, but more and more studies point
out mitochondria as the major organelle regulating the
molecular mechanisms of cellular ageing (Duckles et al.
2007; Hu et al. 2007; Kinnally and Antonsson 2007;
Rasola and Bernardi 2007).
Evidential support, gathered from studies in both
somatic and germ cells, backs-up the theory of
premature ageing induced by cryopreservation
(Martin et al. 2004, 2007; Paasch et al. 2004a; b;
Grunewald et al. 2005; Wundrich et al. 2006; OrtegaFerrusola et al. 2008). Freezing and thawing of hepatocyte mitochondria permeabilize the inner mitochondrial
membrane (Krahenbuhl et al. 1991). Since cytocrome C
is normally bound to the inner mitochondrial membrane
in association with the anionic phospholipid cardiolipin,
in presence of high ROS levels, cardiolopin looses his
binding anity for cytocrome C (Rasola and Bernardi
2007). In addition, high ROS levels interfere with
mitochondrial Ca++ homeostasis, damages mtDNA,
disrupt the properties of the inner membrane barriers,
alter the maintenance of mitochondrial membrane
potential and provoke a dysfunction of the Krebs cycle.
Finally, all these changes lead to low levels of ATP and

347

the release of pro-apoptotic factors in the cytoplasm.

Experiments with spermatozoa as cell model have
shown that cryopreservation leads to increases in
intracellular Ca++ concentration, loses of mitochondrial membrane potential, lowering of ATP levels and
the release of pro-apoptotic factors in the cytoplasm
(Martin et al. 2005; Feng et al. 2006; Schober et al.
2007; Ortega-Ferrusola et al. 2008). All this evidence
stresses the need of further studies attempting the
development of methods to protect the mitochondria
during cryopreservation (Pena et al. 2003a,b, 2005,
2006; Carra et al. 2004; Soladiki et al. 2005; Stainek
et al. 2005; Grunewald et al. 2006, 2007; Said et al.
2006; Swerdlow 2007), and supports the theory that
surviving post-thaw spermatozoa suer from a premature ageing, and not of cryo-capacitation.
Acknowledgements
The investigations of the authors received nancial support from the
Ministerio de Educacion y Ciencia-FEDER Madrid, Spain, grants,
AGL 2007-60598 (GAN) and BFU-2007-62423(BFI); FORMAS (the
former Swedish Council for Forestry and Agricultural Research,
SJFR), the Swedish Farmers Foundation for Agricultural Research
(SLF), and the Swedish Foundation for Equine Research (SSH),
Stockholm, Sweden.

Author contribution
All the authors have participated in the literature review and in the
correction and revision of the manuscript

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Submitted: 16 May 2008

Authors address (for correspondence): FJ Pena, Section of Reproduction and Obstetrics, Department of Herd Health and Medicine,
Faculty of Veterinary Medicine, Avd de la Universidad, s n 10071
Caceres, Spain. E-mail: fjuanpvega@unex.es

 2008 The Authors. Journal compilation  2008 Blackwell Verlag