Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.

Abstract

Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review was to identify and discuss common themes in mitochondrial function related to mammalian reproduction.
The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis, sperm, oocytes, early embryos and embryonic stem cells.
ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochondria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete, embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation into distinct cellular fates.
Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the several systems reviewed. Low mitochondrial activity seems a feature of 'stemness', being described in spermatogonia, early embryo, inner cell mass cells and embryonic stem cells.

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† Introduction
† Search method
† Testis mitochondria
† Sperm mitochondria
† Oocyte/embryo mitochondria
† Mitochondria in embryonic stem cells
† Conclusions
Mitochondrial functionality in
reproduction: from gonads and
gametes to embryos and embryonic
stem cells
Joa ˜o Ramalho-Santos1, Sandra Varum, Sandra Amaral, Paula C. Mota,
Ana Paula Sousa, and Alexandra Amaral
Center for Neuroscience and Cell Biology, Department of Zoology, Faculty of Sciences and Technology, University of Coimbra 3004-517,
Coimbra, Portugal
1Correspondence address. Tel: þ351-239-855-760; Fax: þ351-239-855-789; E-mail: jramalho@ci.uc.pt
table of contents
background: Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium
signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review
was to identify and discuss common themes in mitochondrial function related to mammalian reproduction.
methods: The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis,
sperm, oocytes, early embryos and embryonic stem cells.
results: ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from
mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either
reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochon-
dria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based
calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered
apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete,
embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected
phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation
into distinct cellular fates.
conclusions: Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the
several systems reviewed. Low mitochondrial activity seems a feature of ‘stemness’, being described in spermatogonia, early embryo, inner
cell mass cells and embryonic stem cells.
Key words: mitochondria / testis / sperm / oocyte / embryonic stem cells
& The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Human Reproduction Update, Vol.15, No.5 pp. 553–572, 2009
Advanced Access publication on May 4, 2009doi:10.1093/humupd/dmp016

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Introduction
Mitochondria are double-membrane organelles that play a fundamental
role in the cell, and mitochondrial dysfunction has been linked with
several pathologies, including infertility (reviewed in Wallace, 1999).
Although sharing a general pattern, mitochondria can have distinct fea-
tures, based on inner membrane invaginations (cristae) and matrix struc-
ture. Depending on the cell type and functional status, mitochondria
present an extensive range of morphologies, are functionally hetero-
geneous(Collinsetal.,2002),andvaryinnumber(reviewedinMeinhardt
et al., 1999). ATP synthesis by oxidative phosphorylation (OXPHOS) is
the primary function associated with mitochondria. During this process
electrons derived from the oxidation of substrates are led through
redox carriers of the inner membrane electron transfer chain (ETC) to
the final acceptor, molecular oxygen (O2). Electron transfer is associated
withprotonpumpingintotheintermembranespaceatcomplexesI,IIIand
IV. This establishes an Hþ-based electric and chemical gradient (Dc and
DpH, respectively), then used to drive ATP synthesis via complex V
(ATP synthase) (Fig. 1) (Darley-Usmar et al., 1987; Harris, 1995).
OXPHOS is the most efficient way to oxidize substrates; however,
glycolyticenzymesareevolutionarilyolderandhavereachedcatalyticper-
fection,thussuggestingthatglycolysismaybethepathwayofchoiceifgly-
colytic substrates are plentiful and oxygen is low.
ATP production can vary to match energy demands, and mitochon-
dria are heterogeneous in different tissues. Long-term adaptations to
various rates of ATP utilization can be achieved by modifying the
number, morphology and location of mitochondria, as well as the pro-
portions of certain ETC constituents (Meinhardt et al., 1999; Nogueira
et al., 2001). Two morphologic states have been defined in mitochon-
dria: in the orthodox state cristae tend to be tubes or short flat lamel-
lae with few junctions in the peripheral region of the inner membrane,
although condensed mitochondria have larger internal compartments
with multiple tubular connections (reviewed in Mannella, 2006a;
Fig. 2). These states are interchangeable, and seem to be related to
mitochondrial status, with mitochondria displaying condensed confor-
mation when ADP is in excess but reverting to the orthodox state
when ADP is limiting (Mannella, 2006b). However, changes in mito-
chondrial architecture may also merely reflect osmotic changes in
the local environment (Mannella, 2008).
Mitochondria are also characterized by having their own, maternally
inherited genome (Giles et al., 1980), mitochondrial DNA (mtDNA).
Human mtDNA is a double stranded circular molecule encoding 13
polypeptides that are essential subunits of ETC complexes, 22
tRNAs and 2 rRNAs, that are necessary for their translation
(Anderson et al., 1981). The maintenance and expression of the
mitochondrial genome is controlled by nuclear-encoded factors that
are translocated to the mitochondria (St John et al., 2007).
All in all, 85–90% of a cell’s oxygen is consumed by mitochondria in
OXPHOS. However, this comes with an undesirable extra, the pro-
duction of potentially harmful reactive oxygen species (ROS). Mito-
chondria are the major ROS generator, with 0.2–2% of the oxygen
taken up by the cells converted to ROS by mitochondria (reviewed
in Harper et al., 2004; Orrenius et al., 2007). At several sites along
the ETC (namely complexes I and III) electrons can react directly
with oxygen or other electron acceptors, and generate free radicals
(Muller et al., 2004; Grivennikova and Vinogradov, 2006; Fig. 1). As
a result, mitochondria need continuous protection, provided by
various low-molecular-weight antioxidants, as well as by multiple enzy-
matic defence systems (for review see Ott et al., 2007). However,
recent evidence also highlights a specific role of ROS in cell signalling
(reviewed in Orrenius et al., 2007). Nonetheless, mitochondrial
ROS production is very sensitive to the proton-motive force. As
their name indicates, UCPs uncouple proton flux through the inner
mitochondrial membranes and ATP synthesis, providing a route for
proton re-entry (Mattiasson and Sullivan, 2006) (Fig. 1), lowering
the proton-motive force and attenuating mitochondrial ROS pro-
duction (Brand and Esteves, 2005).
Another major role is mitochondrial participation in apoptosis
(namely via the ‘intrinsic’ apoptosis pathway). A wide range of stress
stimuli can be transduced to mitochondria, resulting in increased per-
meability to mitochondrial proteins such as cytochrome c, which plays
a prominent role in promoting the caspase cascade of cell execution
(reviewed in Khosravi-Far and Esposti, 2004). Once released to the
cytoplasm, cytochrome c triggers a series of events leading to the pro-
teolytic activation of executioner caspases 3, 6 and 7 (reviewed in
Orrenius et al., 2007). Several factors can regulate mitochondria-
mediated apoptosis. Notably, Bcl-2 family members may either
Figure 1 Roles of mitochondria.
(A) The electron transfer chain (ETC), of the inner mitochondrial membrane
involved in oxidative phosphorylation (OXPHOS) and reactive oxygen special
(ROS) production, including ETC complexes (I, II, III, IV and V), electron car-
riers ubiquinone (Q) and cytochrome c (cyct) and uncoupling proteins (UCP).
(B) Integration of mitochondrial functions, including ATP and ROS production,
activation of apoptosis and effects of oxidative stress.
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promote cell survival (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1/Bfl-1) or lead to
cell death (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok) interacting to
form homo- and heterodimers, their relative abundance being the
determinant of the threshold of apoptosis (Zamzami et al.,1998).
The assessment of mitochondrial functionality can be carried out
using oxygen and TPPþ(tetraphenyl phosphonium) electrodes to
analyse respiration and electric membrane potential, respectively.
The fluorescent probe Calcium Green can be used to access
calcium buffering capacity (Amaral et al., 2009), and a range of cationic
fluorescent probes, which accumulate in either isolated or cellular
mitochondria depending on mitochondrial membrane potential
(MMP or DC), although controls must always be carried out to
ensure proper functional measurements (Ramalho-Santos et al.,
2007). Loss of mitochondrial function can be mimicked in vitro by
means of specific drugs that either inhibit the ETC or eliminate the
mandatory link between the respiratory chain and the phosphorylation
system (OXPHOS uncouplers) (Table I).
Search method
A computerized literature search was conducted using Medline and
Web of Knowledge for aspects of mitochondrial function in testis,
sperm, oocytes, early embryos and embryonic stem cells (ESCs). Mito-
chondrial morphology, mitochondrial localization,membrane potential,
electron transfer complex activity, ATP synthesis, ROS production,
antioxidant defences, calcium signalling and apoptosis were the
aspects searched for. Given the controversial issue concerning the
origin of ATP for different steps of gametogenesis, sperm motility
and embryo development, other metabolic pathways were also inves-
tigated in these cases. mtDNA was not included as a primary search
Figure 2 Testis mitochondria.
(A) Different types of mitochondria present in male germ cells. See text for discussion. (B) Electron microscopy of rat testicular mitochondria highlighting the hetero-
geneous mix of mitochondrial types shown in A. Arrow highlights a cross-section typical of the sperm axoneme microtubule organization.
........................................................................................
ETC inhibiton Amytal, Rotenone
Atpenins, TIFA
Antimycin A,
Myxothiazol
Azide, Carbon
monoxide, Cyanide
Oligomycin, DCCD
OXPHOS
uncouplingDinitrophenol
Valinomycin
Nigericin
Krebs cycle
inhibition
Table I Mitochondrial inhibitors and their effects
EffectDrugMode of action
Blocks Complex I
Blocks Complex II
Blocks Complex III
Blocks Complex IV
Blocks Complex V
Hydrophobic proton carriersCCCP, FCCP,
Kþ ionophore
Kþ/Hþ mobile carrier
Succinate analog; Inhibits
succinate dehydrogenase
(Complex II)
Blocks ANT
Malonate
Transport
inhibiton
Ca2þuptake
inhibition
Atractyloside
Ruthenium redInhibits the mitochondrial Ca2þ
uniporter
CCCP, Carbonyl cyanide m-chloro phenyl hydrazone; DCCD,
Diclychlohexylcarbodiimide; FCCP, Carbonyl cyanide-p-trifluoromethoxyphenol
hydrozone; TIFA, 2-Thenoyltrifluoroacetate; ANT, Adenine nucleotide translocator.
Mitochondrial activity in reproduction
555

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criteria, given that it merits a lengthy separate discussion, beyond the
scope of the current review, although some aspects are briefly included
wheretheypertaintootheraspectsofmitochondrialfunction.Relevant
literature was selected to determine common themes throughout the
reproductive system. Given the volume of literature obtained review
articles are cited for general or less controversial topics.
Testis mitochondria
Morphology
Morphology, localization and energy metabolism of testicular mito-
chondria change markedly during spermatogenesis, and three types
of mitochondria are recognizable: orthodox-type mitochondria in
Sertoli cells, spermatogonia and preleptotene and leptotene sperma-
tocytes; the intermediate form in zygotene spermatocytes; and the
condensed form in pachytene and secondary spermatocytes and
early spermatids, a conformation that shifts back to the intermediate
form in late spermatids and spermatozoa (De Martino et al., 1979)
(Fig. 2). An association between germ cell mitochondrial morphology
and metabolic status during spermatogenesis was postulated, in which
the condensed form presents higher efficiency (De Martino et al.,
1979). These morphological changes may be supported/induced by
factors released by Sertoli cells. In fact, Activin A was described as
an inducer of the condensed form, which may be one of the factors
contributing to the regulation of germ cell differentiation by Sertoli
cells (Meinhardt et al., 2000).
Leydig cell mitochondria present lamellar cristae in close associ-
ation, with a gap between apposing lamellae of ?4 nm, a unique
feature of steroid-producing cells. Although the functional significance
of these structures is unknown, it has been suggested that they are not
involved in ATP production since the close apposition of membranes
does not allow for the presence of ATP synthase (Prince, 2002).
Testis-specific components
Concomitant with the described structural changes, several mitochon-
drial proteins, such as heat shock protein (hsp) 60 and 70, Lon pro-
tease and sulphidryl oxidase (SOx), are known to be differentially
expressed during distinct phases of spermatogenesis (for review see
Meinhardt et al., 1999). Specific isoforms are also found in testicular
mitochondria, such as cytochrome c and subunit VIb-2 of the cyto-
chrome c oxidase (COX) (Hess et al., 1993; Hu ¨ttemann et al.,
2003). Finally other proteins are preferentially expressed in the
testis (see Table II). Mutations on the mitochondrial ‘Drosophila’
.............................................................................................................................................................................................
Mt-Hsp60Expressed in Sertoli and Leydig cells,
spermatogonia and early spermatocytes
Table II Mitochondrial testicular proteins and their effects on reproduction
ProteinUnique featuresFunctionReference
Post translational maturation, translocation of
polypeptides, binding of unfolded or partially folded
proteins
Proteolytic ATP-dependent enzyme involved in
mtDNA integrity and degradation of misfolded
proteins (in bacteria)
Oxidation of sulphydryl compounds (ex: glutathione,
cystein and thioglycerol)
Same functions as somatic cytochrome c but with
enhanced anti H2O2capability and higher
pro-apoptotic activity
Electron transport
Reviewed in
Meinhardt et al.
(1999)
Seitz et al. (1995)Mt LON proteasesOnly expressed in the mitochondrial matrix of
early meiotic cells, such as leptotene and zygotene
spermatocytes
Only expressed in pachytene and early round
spermatids
Starts to be expressed in zygotene spermatocytes
slowly taking the place of somatic cytochrome c
mt SOx Seitz et al. (1995)
Testicular cytochrome
c isoform
Liu et al. (2006), Hess
et al. (1993)
Subunit Vib-2-of the
COX
SCaMC-3-Like
Testicular isoform of COX subunit Vib-2 Hu ¨ttemann et al.
(2003)
Traba et al. (2008)
Lacks the Ca2þ-binding N-extension ATP-Mg/Pi exchange (catalyze the net accumulation or
depletion of mitochondrial adenine nucleotides)
Encoded by mitochondrial genome, may reflect higher
energy requirements of meiotic cells
ADP/ATP translocases (involved in OXPHOS)
Atypical dual specificity phosphatases
COXII High levels of expression in pachytene
spermatocytes
Saunders et al. (1993)
AACA/slc25a31
DSP21
Traba et al. (2008)
Rardin et al. (2008)Peripheral membrane protein of the inner
mitochondrial membrane
Expressed at high levels in the testis and at much
lower levels in all other tissues
Compensates for the loss of expression the
somatic form (PDH1) associated with the x
chromosome
mf Ft expression appears correlate with
management of ATP burst in a short time
Highest level of expression in pubertal and adult
testis, in particularly by the haploid germ cells and
Leydig cells
GPAT2Synthesis of triacylglycerol and all glycerophospholipids Wang et al., 2007
PDH2Pyruvate dehydrogenaseKorotchkina et al.
(2006)
Mt FerritinHighly expressed in human testis, particularly in
spermatocytes and Leydig cells
Associated with the mitochondrial inner membrane
that induces mitochondrial fission
Santambrogio et al.
(2007)
Monticone et al.
(2007)
Mitochondrial fission
regulator 1 (Mtfr1)
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protein Merlin, common to somatic cells (ortholog in humans is
‘Neurofibromatosis’), produces viable but sterile males, indicating
that the induced deregulation on mitochondrial function, although
not affecting somatic cells, has profound implications on germ cell
differentiation (Dorogova et al., 2008). However, this is certainly not
a general effect, as mice lacking a testis-specific translocase (Tom
34b) are normal and fertile (Terada et al., 2003).
Leydig cell steroidogenesis
Leydig cell steroidogenesis is reliant on mitochondrial functionality as
demonstrated using the MA 10 Leydig cell line. Results clearly
showed that Dc, ATP synthesis, DpH and mitochondrial [Ca2þ] are
all required for steroid biosynthesis (Hales et al., 2005). Myxothiazol
(a complex III blocker) inhibits LH-stimulated testosterone production
in multiple sites along the steroidogenic pathway (Midzak et al., 2007).
In addition Leydig cell mitochondria are influenced by ROS, notably
during the cholesterol transfer step (Stocco et al., 1993). These
ROS are also used by macrophages as mediators to modulate
Leydig cell activity (Hales, 2002).
Testicular energy metabolism
Testicular mitochondria have different bioenergetic parameters when
compared with mitochondria harvested from other tissues. Specifi-
cally, testis mitochondria are shown to consume less oxygen to gen-
erate approximately the same maximum electric potential as other
tissues, and show an age-related modification in phosphorylative effi-
ciency with young animals presenting less efficient phosphorylation
(Mota et al., 2009; Amaral et al., 2009). These observations suggest
that, contrary to previous studies, testicular mitochondria should be
considered as the primary mitochondrial toxicological model to test
the effect of distinct substances on male gametogenesis (Tavares
et al., 2009).
Testis-specific morphogenetic events suggest that male gonads have
a higher energy requirement than ovaries, starting early at the time of
Sry activation (Matoba et al., 2008). Because spermatogonial stem cells
(SSCs) are slow-dividing, it is expected that low MMP might be a
shared characteristic with other stem cells (see below). The
neonate (0–5 days post-partum (dpp)) rat testis cell fraction with
the highest concentration of SSCs includes gonocytes, and exhibits
low DC, although stem cells in rat pup (8–14 dpp) testes appear to
have more active mitochondria than their gonocyte precursors,
which might reflect increased proliferative activity as this population
expands to fill the rapidly increasing number of SSC niches (Ryu
et al., 2004).
In the adult testis the survival of germ cells is dependent on carbo-
hydrate metabolism, including both anaerobic (glycolysis) and aerobic
(OXPHOS) pathways. However, different cell types differ in their pre-
ferred substrates (Robinson and Fritz, 1981; Grootegoed et al., 1984;
Nakamura et al., 1984; Bajpai et al., 1998; Meinhardt et al., 1999). In
fact, the formation of the blood-testis barrier and the alteration in the
surrounding medium, cause a considerable change in the energy
metabolism of germ cells. Spermatogonia in the basal compartment
are supplied exclusively by blood components. However, after
passage to the luminal compartment germ cells rely on the breakdown
of lactate and pyruvate provided by Sertoli cells (reviewed in
Boussouar and Benhamed, 2004). Therefore, spermatogonia, mature
sperm and the somatic Sertoli cells exhibit high glycolytic activity,
whereas spermatocytes and spermatids produce ATP mainly by
OXPHOS (Robinson and Fritz, 1981; Grootegoed et al., 1984;
Nakamura et al., 1984; Bajpai et al., 1998; Meinhardt et al., 1999).
This could also be a matter of opportunity: since seminiferous
tubule fluid is rich in lactate and poor in glucose, it is hypothesized
that, even though spermatocytes have the machinery to produce
energy through glycolysis, they rely mostly on lactate (Bajpai et al.,
1998). Nonetheless, there are incongruities between availability and
usability. Blood vessels, located exclusively between tubules, supply
the oxygen needed to perform OXPHOS that only reaches the
lumen of the seminiferous tubules by diffusion (Wenger and
Katschinski, 2005). The facilitated access of spermatogonia to
oxygen would lead us to expect the use of OXPHOS, instead of gly-
colysis. Similarly, having less access to oxygen, spermatocytes were
expected to perform glycolysis. However, the substrate availability
imposed by seminiferous tubules compartmentalization, together
with ATP demand, may prime the cell to different adaptations. It is
also possible that stem cells maintain a low metabolism to avoid ROS-
related damage.
Mitochondria-related apoptosis in the testis
In normal spermatogenesis not all germ cells reach maturity, and the
normal physiological death of germ cells via apoptosis seems to be a
constant event which can be potentiated by various stimuli (reviewed
in Sinha-Hikim et al., 2003). Caspases are not only effectors of the
apoptotic process but can induce its activation through the mitochon-
drial pathway. Caspase 2 expression is increased in 16 dpp rat testis,
when germ cell apoptosis also peaks. The increased amounts of acti-
vated caspase 2 in mitochondria was matched by an increased level of
cytochrome c release. Specific inhibitors of caspase 2 mitigate the
increased cytochrome c release, indicating an important upstream
role of mitochondria in germ cell apoptosis during the first wave of
spermatogenesis (Zheng et al., 2006).
Bcl-2 family members are also crucial during the first spermatogenic
wave (Rodriguez et al., 1997). Bax is required to induce germ cell
death in dividing spermatogonia, at the time point at which their
number is regulated in a density-dependent manner (Russell et al.,
2002), as shown by infertile Bax KO mice that present accumulation
of atypical premeiotic germ cells but no mature haploid sperm
(Knudson et al., 1995). In contrast, male bcl-w-deficient mice display
normal testicular development before puberty, although after
puberty Sertoli and germ cells of all types are severely reduced in
number, and seminiferous tubules contain many apoptotic cells and
no mature sperm (Yan et al., 2000). In a different study using cynomo-
logus monkeys (Macaca fascicularis), hormone deprivation, heat, or
both, led to an increase in Bcl-2 levels in testicular lysates and
increased cytochrome c and Smac/Diablo release (Jia et al., 2007).
In mice and humans, hormone deprivation and heat-induced male
germ cell death, also induced the mitochondria-dependent apoptotic
pathway (Vera et al., 2004, 2006). However, recent research
described activation of both intrinsic and extrinsic (Fas-mediated)
apoptotic pathways in situations of FSH and testosterone depletion
in rats and mice (Pareek et al., 2007).
Interestingly, inhibition of ATP synthase decreased ATP levels and
suppressed cell death, an effect not seen with inhibition of glycolysis,
Mitochondrial activity in reproduction
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indicating that mitochondrial ATP production plays a role in regulating
male germ cell apoptosis (Erkkila et al., 2006). In a different study,
apoptosis was triggered by incubating segments of seminiferous
tubules without survival factors (i.e. serum or hormones), and apop-
tosis in spermatocytes and spermatids was significantly repressed at
low levels of oxygen or by inhibitors of mitochondrial transitory per-
meability, revealing another aspect of mitochondrial function in apop-
tosis (Erkkila et al., 1999). Finally, germ cell mitochondria present a
unique feature in apoptosis: testicular cytochrome c isoform shows
a 3–5-fold greater apoptotic activity than the somatic isoform (Liu
et al., 2006). Interestingly, testicular cytochrome c KO mice are
fertile but present with highly atrophied testes, with a reduced
number of spermatocytes, spermatids and spermatozoa (Narisawa
et al., 2002).
Testicular ROS and antioxidant defences
Because mitochondria are the major source of intracellular ROS they
need constant protection from these species, accomplished through a
wide network of mitochondrial non-enzymatic and enzymatic antiox-
idant defences (reviewed in Ott et al., 2007). Thioredoxin glutathione
reductase, a redox enzyme, accumulates in testes after puberty, pri-
marily in elongating spermatids at the site of the mitochondrial
sheath formation (Su et al., 2005). Previously, the same pattern of dis-
tribution had been described for glutathione-peroxidase 4 (GPX4)
(Roveri et al., 1992). Interestingly, prolonged expression of this
enzyme during the spermatogenic cycle (observed in transgenic mice
bearing the rat GPX4) revealed a number of spermatogenic defects
including primary spermatocyte apoptosis, haploid cell loss, seminifer-
ous epithelium disorganization and reduced fertility (Puglisi et al.,
2007). In agreement with this result, high levels of manganese super-
oxide dismutase (SOD) were associated with small testis, male infer-
tility, and decreased female fertility (Raineri et al., 2001). Results
obtained in these studies suggest that any deregulation, or either
loss/overexpression of mitochondrial antioxidant enzymes, disrupt
normal homeostasis of the seminiferous epithelium resulting in
reduced fertility.
Pathological effects of aging on testicular
mitochondria
Cellular ageing has been linked to increased ROS production and
mitochondrial dysfunction (Harman, 1983; Jonhson et al., 1999).
Analysis of testis mitochondria has shown differences in ETC com-
plexes and membrane fatty acid composition throughout development
and ageing. In fact, the activity of ETC complexes follows a common
pattern, with an increase during mitosis and first meiosis of germ
cells, and a decrease with ageing. These changes were correlated
with a drop in polyunsaturated fatty acid content, increased pro-
duction of superoxide and reduced SOD activity (Vazquez-Memije
et al., 2005, 2008). In fact, the balance between pro and antioxidant
agents in the ageing testis is shifted towards pro-oxidation (Rebrin
et al., 2003). Using domestic cat testicular mitochondria it was recently
shown that membrane potential reached a maximum in animals with
fully active reproductive function, after a pubertal period of increasing
values of mitochondrial electric potential (Mota et al., 2009). In the rat,
there was a peak of functionality in adult animals, a decrease with age,
was coupled with an increase in expression and activity of uncoupling
protein 2 (UCP-2), suggesting that proton leakage may have a protec-
tive role in managing age-dependent mitochondrial dysfunction
(Amaral et al., 2008). Mitochondria from Leydig cells also present
alterations with age, consistent with the proposal that mitochondria-
derived ROS may play a role in the decline in testosterone production
(reviewed in Zirkin and Chen, 2000).
Sperm mitochondria
During the differentiation of spermatids into sperm (spermiogenesis),
some mitochondria, like much of the cytoplasm, are lost in the
so-called residual bodies, whilst those remaining rearrange in
elongated tubular structures (Ho and Wey, 2007) and are packed heli-
cally around the anterior portion of the flagellum. Concomitantly, the
number of mtDNA molecules per haploid genome is reduced (Hecht
et al., 1984), which is probably mediated by the down-regulation of
mitochondrial transcription factor A (TFAM) (Larsson et al., 1996,
1997). Once the process is concluded mature mammalian sperm
possess 22–75 mitochondria arranged end to end in the midpiece
(Otani et al., 1988). The anchorage of the mitochondrial sheath to
the axoneme is supported by the sub-mitochondrial reticulum, a
complex of filaments that seems to sustain mitochondrial organization
(Olson and Winfrey, 1990). Furthermore, the outer membranes of
sperm mitochondria are enclosed in a keratinous structure, the mito-
chondrial capsule, formed by disulfide bonds between cysteine- and
proline-rich selenoproteins, including the sperm-specific phospholipid
hydroperoxidase glutathione peroxidase (Ursini et al., 1999). This
structure appears to confer mechanical stability, and is responsible
for some distinctive features of sperm mitochondria, namely the resist-
ance to hypo-osmotic stress, and the unfeasibility of completely isolat-
ing these organelles. The fact that some mitochondria are
evolutionarily retained in a very specialized sperm region suggests
that these organelles fulfil a crucial role in sperm function. However,
their physiological significance is still unclear.
Mitochondrial activity and sperm function
Mitochondria may supply sperm with energy for several purposes,
including sperm motility. Electron microscopy revealed that sperm
from asthenozoospermic samples have disordered mitochondria, sig-
nificantly shorter midpieces and fewer mitochondrial gyres than nor-
mozoospermic counterparts, whereas midpiece widths and tail
lengths were similar (Mundy et al., 1995). Moreover, enzymatic activi-
ties of ETC complexes are positively correlated with human sperm
quality, particularly motility (Ruiz-Pesini et al., 1998, 2000b). Likewise,
the expression of ETC subunits is associated with human sperm
quality (Amaral et al., 2007).
The relevance of mitochondrial activity in sperm function has been
studied at the gene level, namely the significance of sperm mtDNA
integrity in male (in)fertility. Although some conflicting results have
been published on the effects of specific mitochondrial point mutations
and large-scale deletions (for review see St John et al., 2007), it seems
consensualthattheaccumulationofmultiplemtDNArearrangementsis
associated with loss of sperm function (St. John et al., 2001). Similarly,
boththenumberandsizeofmtDNAdeletionsineithertesticularoreja-
culated sperm are negatively correlated with ICSI pregnancy outcomes
(Lewis et al., 2004), clearly showing the deleterious effect of mtDNA
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rearrangements, even when low motility is bypassed. These outcomes
have also been supported by transmitochondrial mice models, where
the accumulation of pathogenic mtDNA-derived ETC defects was
responsible for male infertility (Nakada et al., 2006). Furthermore,
certain mtDNA haplogroups (groups of specific mtDNA types) have
been suggested to predispose for reduced sperm motility (Ruiz-Pesini
et al., 2000a; Montiel-Sosa et al., 2006), although this has been contra-
dicted by others (Pereira et al., 2005). On the other hand, a number of
reports have suggested that low quality sperm present abnormal
mtDNA content (May-Panloup et al., 2003; Amaral et al., 2007), and
theexpressionofTFAMandofthecatalyticsubunitofDNApolymerase
gamma (POLG), both of which are implicated in the regulation of
mtDNA copy number, are both lower in poorer quality sperm
(Amaral et al., 2007). The relevance of POLG in male infertility has
also been confirmed using mouse models: homozygous knock-in mice
expressing a deficient POLG presented increased levels of mtDNA
mutations and deletions, and showed reduced lifespan and premature
onsetofaging-relatedphenotypes,includingreducedfertility(Trifunovic
et al., 2004). In addition, polymorphisms in the CAG-repeat region of
POLG are possibly associated with sperm quality, although their true
significance in male infertility is questionable (Amaral et al., 2007).
Overall, anddespitethe controversiesonthe exacteffectof aparticular
mtDNA point mutation, deletion or haplogroup in human sperm, there
is clear evidence that alterations in the mitochondrial genome can com-
promise sperm motility and function.
The link between mitochondria-generated ATP and sperm motility/
fertilization competence has been illustrated by Dc evaluation (Fig. 3).
Several studies confirmed that motility is related to mitochondrial
functional status in humans (Troiano et al., 1998), equines
(Loveet al., 2003), rats (Gravance et al., 2001), boars (Spinaci et al.,
2005) and rams (Martinez-Pastor et al., 2004). Additionally, at least
in humans, sperm fertilization potential (measured as fertilization
rates after IVF) is strongly related to Dc and thus to mitochondrial
functionality (Kasai et al., 2002; Marchetti et al., 2002, 2004b). On
the other hand, Dc seems to negatively correlate with both DNA frag-
mentation and the generation of ROS (Wang et al., 2003). Mirroring
the importance of mitochondria to sperm function, subpopulations
of mitochondria with high Dc are enriched in cells with elevated
fertilization capabilities (Gallon et al., 2006). Interestingly, it has
recently been proposed that the ultimate cause for the negative
effect of endocannabinoids in male reproduction may be the reduction
of sperm mitochondrial activity (Rossato, 2008). These cumulative
outcomes strongly suggest Dc as an indicator of sperm functional
status.
The role of sperm mitochondrial metabolism
is controversial
Sperm require ATP mainly for motility, as well as for the cellular events
involved in hyperactivation, capacitation and the acrosome reaction.
The provenance of the ATP that fuels these events, especially motility,
has been discussed for decades: is it derived from OXPHOS, or purely
glycolytic? The debate has been confounded by possible species-
specific discrepancies, as well as by differences in experimental con-
ditions. The usual approach is to analyse motility and/or ATP levels
after incubating sperm in media containing different substrates, in
the presence or absence of inhibitors. However, media composition,
temperatures and times of incubation, as well as the inhibitors and
their concentrations, vary from study to study.
Thecompartmentalizationofmitochondriainthespermmidpiecemay
limit the availability of OXPHOS-derived ATP for the dynein-ATPases
located in the principal piece. This is especially true in species with long
sperm tails, such as rodents, as it is doubtful that sufficient ATP can
diffuse to the distal end of the flagellum. This problem could be solved
via ATP shuttles, but their significance in mammals needs further confir-
mation(Ford,2006). Alternatively, ATPcanbeproducedinthe principal
piecethroughtheglycolyticpathway.Indeed,glycolyticenzymesseemto
becompartmentalizedinthefibroussheath,acytoskeletalelementofthe
principal piece (Krisfalusi et al., 2006; Kim et al., 2007).
Elegant work was done using hypotonically treated cells, i.e. with
disrupted membranes, to solve the difficulty in isolating sperm mito-
chondria and then analysing oxygen consumption and respiration
rates with various substrates (Keyhani and Storey, 1973; Storey,
1978; Carey et al., 1981). Results were species-specific, i.e. sperm
from distinct species have dissimilar capabilities to metabolize different
substrates, suggesting that mitochondrial activity in sperm of a given
species is adjusted to the substrate content of the female tract
(Storey, 1980).
Human sperm motility and ATP content can be maintained in the
presence of a glycolyzable substrate, but decline rapidly in its
absence, even in the presence of oxidizable substrates (Peterson and
Freund, 1970; Williams and Ford, 2001). On the other hand, the pre-
viously stated fact that mutations in the mitochondrial genome are
associated with decreased sperm quality suggests that OXPHOS is
also relevant. Interesting outcomes were obtained from a patient har-
bouring a mtDNA point mutation causing reduced activity of the ETC
complex I, and whose sperm presented low-motility. Supplementation
with succinate, a substrate for complex II, circumventing the effect of
the mutation, resulted in an increase in sperm motility (Folgero et al.,
1993). Furthermore, incubation of human sperm with distinct ETC
inhibitors in media containing glucose, resulted in rapid decreases in
sperm motility (Ruiz-Pesini et al., 2000a; St John et al., 2005a, b).
Experiments in rat sperm have shown that 6-chloro-6-deoxysugars
prevent glucose metabolism, and have contraceptive action (Ford
et al., 1981a, b). Sperm became immotile and presented low ATP
Figure 3 Mitochondria in human sperm.
(A) Mitochondrial membrane potential (MMP) detection in live human sperm
using Mitotracker Red (red), nuclear DNA is counterstained with SYBR 14
(green). (B) Immunodetection of a subunit of COX, part of the electron trans-
fer chain (ETC) (complex IV), in the sperm midpiece (green), nuclear DNA is
counterstained with DAPI (blue).
Mitochondrial activity in reproduction
559

Page 8

levels when glucose was the only substrate available, but no detrimen-
tal effects were observed in the presence of pyruvate and lactate (Ford
and Harrison, 1981a, b). Likewise, sperm from control rats presented
higher motility and ATP levels in medium with pyruvate and lactate.
a-Chlorohydrin has also been used to inhibit glycolysis, in epididymal
ram and ejaculated boar sperm, resulting in decreased motility and
ATP concentration, but only in the presence of glucose (Ford and
Harrison, 1985, 1986). Taken together, these outcomes may indicate
a role of OXPHOS in these species, although a contribution of
glycolysis is also evident. The results of experiments in mouse epididy-
mal sperm also seem to imply that both glycolysis and OXPHOS are
able to sustain sperm motility, although with glycolysis in a predomi-
nant role (Mukai and Okuno, 2004).
In testis-specific cytochrome c homozygous knockout mice, males
were fertile, although presenting early testicular atrophy, as discussed
earlier. Moreover, when compared with wild type, their sperm were
less motile, presented lower ATP content and were less successful
in in vitro fertilization (Narisawa et al., 2002). On the other hand,
the disruption of the spermatogenic cell-specific glycolytic enzyme gly-
ceraldehyde 3-phosphate dehydrogenase-S (GAPDH-S) has also
resulted in some remarkable outcomes (Miki et al., 2004). Male homo-
zygous KO mice were infertile (but females were fertile) and their
sperm showed both decreased motility and ATP levels, although mito-
chondrial activity was unchanged. These results have been interpreted
as proof that glycolysis is essential for sperm motility. However, this
view has been challenged (Ford and Harrison, 2006; Ruiz-Pesini
et al., 2007; Storey, 2008), the reasoning being that the inactivation
of GAPDH-S (similarly to 6-chloro-6-deoxysugars, DOG and
a-chlorohydrin) block the glycolytic net synthesis of ATP, but the gly-
colytic ATP-consuming phase still operates. Often neglected is both
that glycolysis is usually a prerequisite for OXPHOS and that, unlike
OXPHOS, it actually requires ATP to initiate the process, a fact that
must always be taken into consideration. Nevertheless, disruption of
the glycolytic enzyme lactate dehydrogenase-C4(LDH-C4), normally
expressed in spermatogenic cells and also in minor amounts in
oocytes, resulted in decreased sperm function and impairment of
male fertility in mice (Odet et al., 2008). Recently, a new approach
has been developed to assess sperm motility, which relies on the
use of laser tweezers to measure swimming speeds and force
(Nascimento et al., 2006). Unexpectedly, no relationship was found
between either human or dog sperm motility and Dc, also suggesting
a predominant role of glycolysis (Nascimento et al., 2008).
The role of OXPHOS versus glycolysis in other events leading to
fertilization is believed to be species-specific. To this extent, glucose
seems necessary for the acquisition of hyperactivated motility, capaci-
tation and acrosome reaction in both humans and mice (Hoppe, 1976;
Fraser and Quinn, 1981; Rogers and Perreault, 1990), and may partici-
pate in the protein phosphorylation events occurring in rhesus
macaque sperm capacitation (Hung et al., 2008). On the other
hand, glucose seems to inhibit capacitation in bull and guinea pig
(Rogers and Yanagimachi, 1975; Rogers et al., 1979; Parrish et al.,
1989), where oxidizable substrates are required. Also noteworthy
are two recent findings using proteomic approaches. The first study
concerns sperm epididymal maturation in rodent species (Aitken
et al., 2007). Apparently, caput epipydimal sperm possess silent mito-
chondria, although caudal sperm have polarized mitochondria, and
therefore epididymal maturation may involve the activation of sperm
mitochondria, and mitochondria-generated ATP may facilitate the
tyrosine phosphorylation events associated with capacitation. The
second study involves the identification of proteomic differences in
asthenozoospermic samples (Martinez-Heredia et al., 2008). The
authors found 17 proteins expressed at different levels in astheno-
zoospermic samples compared with controls. Interestingly, the list of
proteins includes decreased expression of the ETC enzyme
COXVIb. In contrast, none of the glycolityc enzymes were affected.
These cumulative reports seem to demonstrate that in the few days
it can spend in the female reproductive tract mammalian sperm might
be able to utilize both glycolysis and OXPHOS to produce ATP for
different purposes. The balance between these two metabolic path-
ways may vary between species, according to the substrates available
during the sperm’s route.
Sperm mitochondria and ROS
Although seminal leukocytes were thought to be the only ROS-
generators in an ejaculate, it is now well established that sperm are
also responsible for some ROS production (Aitken et al., 1996).
Accordingly, both seminal plasma and sperm possess a number of
antioxidant strategies to protect the male gametes against ROS
damage. These include enzymes such as SOD, catalase, the gluta-
thione peroxidase/reductase system and non-enzymatic substances,
such as ascorbic acid, glutathione and a-tocopherol, among others
(reviewed by Tremellen, 2008).
Low and regulated levels of ROS have been implicated in sperm
capacitation, acquisition of hyperactivated motility, acrosome reaction
and oocyte interaction (for review see de Lamirande et al., 1997; Ford,
2004). When the physiological equilibrium between ROS production
and scavenging is perturbed, sperm function may be compromised
and indeed oxidative stress is implicated in male infertility.
Increased ROS levels have been associated with sperm lipoperoxida-
tion damage, decreased motility, DNA fragmentation and increased
apoptosis (Agarwal et al., 2008). Importantly, it has been recently
proposed that mitochondria are a major contributor to the oxidative
stress experienced by defective human sperm (Koppers et al., 2008).
Mitochondria-related apoptosis in sperm
Although it is well established that a fraction of sperm in any ejaculate
presents apoptotic markers (e.g. Varum et al., 2007), the mechanisms
involved in putative sperm apoptosis are not completely characterized.
However, and among other mechanisms, apoptosis can be triggered
via the mitochondrial pathway. To this extent, the presence of acti-
vated caspase 3 in ejaculated human sperm midpiece was clearly
demonstrated (Weng et al., 2002), and poorer quality samples exhib-
ited higher levels of active caspase 3 positive-sperm. Furthermore, the
activation of caspase 3 is correlated with the externalization of phos-
phatidylserine (Kotwicka et al., 2008). Similar results were obtained
for caspase 9 which, when expressed, is also localized in the midpiece
(Paasch et al., 2004a). Additionally, the treatment of sperm with betu-
linic acid, an inducer of the intrinsic apoptotic pathway, resulted in loss
of Dc and activation of caspases 3 and 9, with a concomitant decrease
in sperm motility (Paasch et al., 2004b; Grunewald et al., 2005).
Indeed, caspase 3 activity seems to be negatively correlated with
sperm Dc (Marchetti et al., 2004a). The transcript for the apoptotic
marker Bcl-2 is also present in ejaculated sperm, with higher levels
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in infertile men compared with controls (Steger et al., 2008). Future
studies will be needed to better determine the role of mitochondria
in triggering sperm apoptosis, and its relevance in sperm (dys)function.
Other possible roles of sperm mitochondria
The involvement of Ca2þsignalling in the regulation of several aspects
of mammalian sperm function is very well documented (Felix, 2005;
Publicover et al., 2008). On the other hand, the ability of mitochondria
to store Ca2þhas been demonstrated in sperm from diverse species,
but its role is unclear (Publicover et al., 2007). Thus, although mamma-
lian sperm mitochondria are able to function as intracellular Ca2þ
stores, a clear role in signalling has not been demonstrated.
Another issue relates to protein synthesis. It is generally accepted
that gene expression in mature sperm is restricted to the mitochon-
dria. In fact, mammalian sperm seem to be able to synthesize both
mitochondria-encoded RNAs (MacLaughlin and Terner, 1973; Hecht
and Williams, 1978; Alcivar et al., 1989) and proteins (Ahmed et al.,
1984; Twaina-Bechor and Bartoov, 1994). More recently, however,
and in contrast with previous data (Diez-Sanchez et al., 2003), mam-
malian sperm were suggested to synthesize nuclear-encoded proteins,
at least during capacitation (Gur and Breitbart, 2006). The results are
particularly odd, and certainly require independent confirmation, not
only because they seem to contradict the dogma that sperm are trans-
lationally silent cells (at least for nuclear-encoded proteins), but also
because they also suggest that translation of nuclear-encoded proteins
occurs in mitochondria-type ribosomes localized either inside or
outside mitochondria, but with no involvement of the cytoplasmic
translation machinery, an event with no equivalent in any other
cell type.
Oocyte/embryo mitochondria
Mitochondria are the most abundant and prominent organelle in the
early embryo (Motta et al., 2000; Sathananthan and Trounson,
2000) and are thought to be exclusively derived from the oocyte
(Cummins, 2000). Contradicting a notion still found in a number of
textbooks, in mammals the entire sperm enters the oocyte at fertiliza-
tion (Ankel-Simons and Cummins, 1996), however, sperm mitochon-
dria are diluted beyond detectable levels or destroyed inside the
embryo (Sutovsky et al., 1999).
Oocyte mitochondrial dysfunction, expressed as declined cell res-
piration and electron transport, may contribute to diminished fertility,
and be the cause of development retardation and arrest in human pre-
implantation embryos (Fissore et al., 2002; Ramalho-Santos et al.,
2004; Thouas et al., 2004). Intracytoplasmatic injection of ‘normal’
mitochondria can overcome mitochondrial dysfunctions (Nagai et al.,
2004) and inhibit oocyte fragmentation (Perez et al., 2000), stressing
the importance of mitochondria in cell death (Fissore et al., 2002).
In contrast, injection of abnormal mitochondria induces oocyte apop-
tosis (Perez et al., 2007).
Mitochondrial number and structure
in the oocyte and early embryo
Oocyte mitochondria must support early embryo development until
the resumption of mitochondrial replication, which only occurs
post-implantation (reviewed in Dumollard et al., 2006). Depending
on the species, a mammalian oocyte contains around 105–108mito-
chondria, with 105in human (Chen et al., 1995; Jansen and de Boer,
1998). Mitochondria propagate from a restricted founder population
present in the primordial germ cell (PGC) (Cummins, 2000; Jansen,
2000), ensuring that mitochondria in the mature oocyte (and there-
fore in dividing blastomeres) are homogeneous. The mtDNA bottle-
neck theory (Hauswirth and Laipis, 1982) suggests a restriction in
the number of mtDNA molecules to be transmitted from the
mother to the offspring, followed by a strong amplification in
oocytes (Reviewed in May-Panloup et al., 2007). The bottleneck
occurs in order to maintain mtDNA homoplasmy and minimize het-
eroplasmy (Cummins, 2001) Therefore, a selection of a group of
mtDNA molecules to repopulate the next generation takes place,
and deleterious mutations tend to be eliminated so as not to be trans-
mitted to the offspring. The nature of the mtDNA bottleneck has
been recently discussed and different mechanisms have been pro-
posed (Cao et al., 2007; Cree et al., 2008; Khrapko, 2008).
However, further investigations are needed to clarify exactly when
and how this phenomenon occurs.
During oogenesis there is an amplification in mitochondrial number
in parallel with cytoplasmatic volume increase. Pre-migratory PGC
have less than 10 mitochondria, 100 mitochondria are present in
ovarian PGCs and 200 in oogonia. Primordial follicle oocytes have
10 000 mitochondria, a number which ultimately increases 10-fold.
In the mature oocyte, each of the 105mitochondria possesses a
single copy of mtDNA (reviewed in Jansen and de Boer, 1998).
The increase in mitochondrial number during oocyte growth is
accompanied by changes in their ultrastructure (Wassarman and
Josefowicz, 1978; Motta et al., 2000; Au et al., 2005). Small oocytes
contain mitochondria with numerous transversely-oriented cristae,
although growing oocytes present round and oval-shaped mitochon-
dria, with columnar-shaped arched cristae. At ovulation, mitochondria
have a spherical immature structure, are highly vacuolated, with a
dense matrix and only few cristae. Between the zygote and the
2-cell stage, mitochondria assume a dumb-bell shape and present
concentrically located cristae. From the 4-cell to the morula
stage, mitochondria have a more elongated structure with transverse
cristae.
The total number of mitochondria in a normal human blastocyst is
about 14 000, and the average number of mitochondria per cell is
about 150 (Van Blerkom, 2008). However, studies in mouse and
hamster models show that the average mitochondria per cell is
higher in the trophectoderm (TE), which will give rise to the placenta,
than in the inner cell mass (ICM), which will give rise to the embryo
proper (Barnett et al., 1996; Van Blerkom, 2008). There is some con-
troversy regarding the morphological homogeneity of the mitochon-
dria found at the blastocyst stage. Although some authors claim that
mitochondria in mouse and human blastocysts are homogenous and
elongated elements (Sathananthan and Trounson, 2000), the existence
of two types of mitochondria in the mouse blastocyst has been
reported: spherical mitochondria in the ICM and elongated mitochon-
dria in the TE. In both types mitochondrial cristae are transversely
oriented and their matrix is less dense than the mitochondrial
matrix found in earlier developmental stages (Stern et al., 1971). Inter-
estingly, although the ICM cells have low Dc and are almost quiescent,
the TE cells are highly polarized and very active producing more ATP
Mitochondrial activity in reproduction
561

Page 10

and consuming more oxygen (Barnett et al., 1996; Houghton, 2006;
Van Blerkom et al., 2006).
Localization and distribution of mitochondria
in the oocyte and early embryo is highly
regulated
During oocyte maturation, and in early embryos, mitochondria are
relocated to different regions, probably in response to localized
energy demands (reviewed in Bavister and Squirrell, 2000). Through-
out maturation mitochondria are mainly found in clusters in close
proximity to endoplasmic reticulum membranes (Jansen, 2000),
suggesting a possible interaction between the two organelles
(Dumollard et al., 2006). In fully-grown germinal vesicle (GV) stage
oocytes mitochondria are concentrated in clusters surrounding the
nucleus (Jansen, 2000; Sun et al., 2001) and migrate to the periphery
of the oocyte after GV breakdown. In metaphase-II (MII) arrested
oocytes, mitochondria are mainly present around the meiotic
spindle and at the oocyte centre (Dumollard et al., 2004, 2006),
accumulating around pronuclei following fertilization, and maintaining
close nuclear association through the morula stage. Impaired redistri-
bution of mitochondria may compromise fertilization and embryo
development (Au et al., 2005), and blastomeres that receive an
insufficient amount of mitochondria remain undivided and undergo
fragmentation (Van Blerkom et al., 2000).
In addition, mitochondrial populations present heterogeneity in
terms of DC. Two populations of mitochondria are present: one
with low DC, which is more abundant, and a smaller amount with
high polarization. Clusters of highly-polarized mitochondria are loca-
lized in the subplasmalemmal/pericortical cytoplasm in oocytes and
early blastomeres (Van Blerkom et al., 2002). Loss of these mitochon-
drial domains affects division (Van Blerkom and Davis, 2006), which
may be associated with the focal ionic and metabolic regulation
(Van Blerkom et al., 2003) involved in oocyte activation and early
development (Van Blerkom et al., 2002; Van Blerkom and Davis,
2007).
Energy metabolism in the oocyte
and early embryo
During oocyte development, a combination of metabolic pathways is
found. Pyruvate and glucose are used by primordial follicles, suggesting
that both OXPHOS and glycolysis are involved (Biggers et al., 1967;
Boland et al., 1993; Wycherley et al., 2005). Furthermore, glucose
used by the cumulus cells may lead to pyruvate production that is uti-
lized by the oocyte (Jansen and Burton, 2004).
Between the primary and pre-ovulatory stages, pyruvate uptake
increases 2-fold (Harris et al., 2008), accompanied by an increase in
O2consumption. However, the average level of ATP seems constant
between the GV and MII stages (Van Blerkom et al., 1995). The
mature oocyte displays a high ATP turnover, supplied by mitochon-
drial respiration (Dumollard et al., 2004) and by the uptake of pyru-
vate (Leese, 1995), which is also the main substrate used by zygotes
(Biggers et al., 1967; Leese and Barton, 1984). At fertilization,
where higher ATP levels are required to support cortical granules exo-
cytosis, chromosome dysjunction, polar body extrusion and Ca2þ
homeostasis (Van Blerkom et al., 1995), there is an increase in O2
use (Magnusson et al., 1977). Therefore, pyruvate is essential for
meiotic maturation and to support the first cleavage division (Biggers
et al., 1967).
From zygote to morula, the levels of ATP and O2used remain basi-
cally constant, and it is essentially substrates for OXPHOS that are
metabolized (Slotte et al., 1990; Van Blerkom et al., 1995). In later
stages the pattern of energy metabolism for the cleaving embryo
changes (reviewed in Dumollard et al., 2007). At the morula stage,
mitochondrial and metabolic changes occur gradually, and a shift in
ATP production to glycolysis is evident (Leese, 1995; Van Blerkom
et al., 1995; Thompson et al., 1996). Glucose is the predominant sub-
strate that supports later embryo development (Biggers et al., 1967;
Gardner and Leese, 1986; Hardy et al., 1989; Gott et al., 1990), but
the increase in glucose uptake at the blastocyst stage is accompanied
by a substantial increment in ATP generation and O2consumption
(Houghton and Leese, 2004), suggesting OXPHOS also takes place
(Dumollard et al., 2007). After implantation, levels of O2use decrease
to those found in pre-blastocyst stages (Houghton and Leese, 2004).
In summary, the uptake of pyruvate is high in the mature oocyte,
drops just after fertilization and then peaks before declining again at
the morula stage (Gardner and Leese, 1986). Interestingly, if one sim-
ultaneously considers independent data for sperm and oocyte, the
building consensus is that the male gamete predominantly uses glycoly-
sis to reach the oocyte, although at the same time the female gamete
is seemingly more reliant on OXPHOS, despite the same substrate
and oxygen availability.
Importantly, intra- and inter-individual variations in oocyte ATP
content have been described, and there is a close association
between oocyte ATP concentration and developmental competence
of the resulting embryo (Van Blerkom et al., 1995). Furthermore, pyr-
uvate and glucose uptake are lower in arrested embryos (Hardy et al.,
1989; Gott et al., 1990), which are also unable to switch to a glucose-
based metabolism when necessary (Gott et al., 1990). Additionally,
blastocysts that implant and develop to term have a significantly
higher glucose uptake prior to transfer than those that fail to
develop (Gardner and Leese, 1987). As in the case of sperm, it is
also possible that these constant changes are simply adjustments to
the substrates available in distinct region of the female reproductive
tract (Jansen and Burton, 2004). Taking cellular volume into
account, Harris and coworkers (2008) found that metabolism is
higher in primary follicles, indicating that energy demands are
greater. On the other hand, a relatively low metabolism is found in
embryos, which seems associated with embryo vitality (Lane and
Gardner, 1996; Leese, 2002).
The relevance of mitochondrial activity in terms of mtDNA has
been also studied. A correlation between mutations in the catalytic
subunit of POLG and premature ovarian failure has been noted and
is probably due to an accumulation of mtDNA deletions as has
been observed for other tissues (Luoma et al., 2004). Female mice
that carry a proofreading-deficient POLG have reduced fertility
(Trifunovic et al., 2004). Furthermore, a relationship between
oocyte mtDNA copy number and oocyte quality/fertility was
observed (Yesodi et al., 2002; Almeida-Santos et al., 2006), and ferti-
lized oocytes present a higher mtDNA copy number than unfertilized
oocytes (Almeida-Santos et al., 2006). This may suggest that it is not
primarily OXPHOS dysfunction that contributes to diminished fertility,
but rather reduced mitochondria/mtDNA copy number that leads to
the OXPHOS dysfunction observed and subsequently to poor quality
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oocytesorreducedfertility(Jacobsetal.,2006).However,contrarytothe
situation in males, females that carry mtDNA with pathogenic mutation
are fertile. These females produce oocytes with a predominant amount
of the mutant mtDNA, oocytes that survive despite severe OXPHOS
defects.ThemutantmtDNAismaternallytransmittedtotheF1–F3pro-
genies, which present mitochondrial dysfunction in various tissues and
have a shorter lifespan due to the consequent pathologies that are unre-
lated to fertility itself (Inoue et al., 2000).
Mitochondria in Ca21signalling
Oocyte mitochondria have an important role in the regulation of
sperm-triggered Ca2þwaves essential for zygote activation (reviewed
in Dumollard et al., 2006, 2007), probably acting as a Ca2þstore and
participating in the generation of the intracellular [Ca2þ] oscillations
(Tesarik and Sousa, 1996; Liu et al., 2001). Sperm-triggered Ca2þ
oscillations stimulate mitochondrial energy production at fertilization,
leading to an increase in O2consumption that is maximal during
Ca2þrelease (Dumollard et al., 2003), and can be primarily initiated
by an influx of Ca2þinto the mitochondria (Dumollard et al., 2006).
Accordingly, the Ca2þsignal is directly transmitted to the mitochondrial
matrix,leadingtotheup-regulationofOXPHOS,whichis,inturn,necess-
ary for the maintenance of [Ca2þ]ioscillations (Dumollard et al., 2004).
Thus, Ca2þlinks ATP supply and demand, allowing for the maintenance
of a low-level of OXPHOS, which increases only when ATP is needed
to support post-fertilization events, stimulated by Ca2þwaves. The pro-
duction of ROS by the ETC is therefore minimized, ensuring that mito-
chondria are exposed to low levels of oxidative stress. Furthermore,
mitochondriaarealsoimportantforcalciumclearance,i.e.inthemainten-
ance of low levels of cytosolic [Ca2þ].
Mitochondrial ROS and apoptosis in oocytes
and embryos
Accumulating evidence has shown that ROS play important roles in
female reproduction (reviewed in Agarwal et al., 2005). However,
the mammalian oocyte and embryo are very sensitive to oxidative
stress (Liu et al., 2000) and if physiological levels of ROS are beneficial,
higher levels can disrupt oocyte maturation and embryo development
(Harvey et al., 2002), and promote embryo fragmentation (Johnson
and Nasr-Esfahani, 1994; Yang et al., 1998).
Indeed, oxidative stress can induce apoptosis of the oocyte and
early embryo (Liu et al., 2000). Mitochondria-dependent apoptosis
seems to be responsible for the post-natal decline in the female
germ cell population (Reynaud and Driancourt, 2000; Tilly, 2001)
and for follicular atresia (Kim and Tilly, 2004), as well as in oocytes
and early embryos (Liu et al., 2000). Mammalian oocytes express
several anti- and pro-apoptotic members of the Bcl-2 family and it is
the balance between these factors that determines oocyte survival
(Liu et al., 2000; reviewed in Jurisicova and Acton, 2004). Further-
more, and mirroring the importance of mitochondria in oocyte apop-
tosis, the apoptotic inducer hydrogen peroxide induces cytochrome c
release from oocyte mitochondria, associated with a decrease in DC
(Liu et al., 2000).
In essence, mature oocytes and early embryos maintain an overall
low-level (i.e. ‘quiet’) metabolism, thus minimizing oxidative stress,
but generating the necessary ATP to fulfil cellular functions (Leese,
2002; Leese et al., 2007).
Physiological alterations associated
with aging
A negative correlation has been described between maternal age and
mitochondrial activity (Jansen and de Boer, 1998). Oocytes from older
women present declining mitochondrial function, which can contribute
to declining fertility, and may be associated with lower embryo devel-
opment and pregnancy rates (Wilding et al., 2001). Ocytes from older
women often present aberrant spindle formation, abnormal chromo-
somal alignment, and consequently, a high occurence of aneuploidy
(Battaglia et al., 1996; reviewed in Eichenlaub-Ritter et al., 2004). It
has been suggested that these abnormalities can be due to an
inadequate capacity to generate sufficient ATP levels to support
these events (Gaulden, 1992). In accordance, oocytes from older
women present an accumulation of mtDNA point mutations (Barritt
et al., 2000) and higher levels of mtDNA deletions (Keefe et al.,
1995), factors that can ultimately be responsible for aneuploidy and
poor implantation rates (Bartmann et al., 2004), ensuring that only
metabolically intact embryos develop to term (Dumollard et al.,
2006). These abnormalities can be corrected by injecting cytoplasm
from younger oocytes (discussed in Klein and Sauer, 2001). The
donor cytoplasm may thus ‘rescue’ spindle misalignments, due, at
least in part, to the mitochondria of the younger oocytes. Moreover,
oocytes from older women present a higher volume fraction of mito-
chondria, indicating compensatory mechanisms (Muller-Hocker et al.,
1996). In the same vein, mitochondrial dysfunction seems to also play
an important role in fragmentation in postovulatory aged oocytes.
Indeed, aged oocytes present a lower uptake of pyruvate (Hardy
et al., 1989). It was also found that, in these oocytes, [Ca2þ] oscil-
lations fail to trigger ATP production and instead induce apoptosis
(Gordo et al., 2002). A decrease in fertility potential, in both maternal
aging and postovulatory aged oocytes, seems to be primarily due to
damages in mitochondria caused by oxidative stress (Fissore et al.,
2002), leading to apoptosis (Perez et al., 1999).
Mitochondria in embryonic stem
cells
ESCs, are derived from the ICM of the blastocyst stage embryo prior
to implantation and can be maintained in vitro in colonies for prolonged
periods without losing the abilities of indefinite self-renewal or differ-
entiation into tissues from all three germ layers (Evans and Kaufman,
1981; Martin, 1981; Thomson et al., 1998; Amit et al., 2000; Pan
and Thomson, 2007). This can be assessed in vitro (through embryoid
body formation) or in vivo (by teratoma formation). Because of these
biological properties, human embryonic stem cells (hESCs) have an
enormous potential as models to study cell differentiation and for
possible replacement cell therapies. Indeed, several groups have
shown that under specific culture conditions hESCs can differentiate
into various somatic cell types (for review see Gepstein, 2002;
Dhara and Stice, 2008; Raikwar and Zavazava, 2009).
MitochondrianumberandmorphologyinESC
As ESCs are derived from the ICM one should expect that they share
metabolic and morphologic features. Indeed, and although there are
line-specific differences, it has been shown that undifferentiated
hESCs have few mitochondria arranged in small perinuclear clusters,
Mitochondrial activity in reproduction
563

Page 12

and immature morphology, evidenced by the presence of few cristae
and low electron lucid matrix (Oh et al., 2005; St John et al., 2005a, b;
Cho et al., 2006) (Fig. 4). hESCs colonies are characterized by high
nuclear cytoplasmatic ratios and cells tightly packed within colonies.
Although there seems to be a paucity of intracellular organization
(sometimes discussed as a ‘stemness’ attribute), this could also be a
reflection of reduced cytoplasm. Furthermore, it is well accepted
that cells in the periphery of the colony are among the first cells to
undergo spontaneous differentiation during in vitro culture. Interest-
ingly these cells have higher levels of mitochondria (Cho et al., 2006).
MMP and metabolism in ESC
There is some controversy regarding the polarization of mitochondria
in undifferentiated versus differentiated ESC. Undifferentiated mouse
ESC has been reported to have highly polarized mitochondria,
which decreases upon differentiation to cardiomyocytes (Chung
et al., 2007). On the other hand, no differences in Dc between undif-
ferentiated hESCs and differentiated hESCs were reported (Saretzki
et al., 2008). The controversy might be due to the fact that these
data come from ESCs of different species, mouse and human, respect-
ively. In addition, mouse ESC were specifically differentiated into car-
diomyocytes, whereas with hESCs spontaneous differentiation was
studied, and as consequence a mixture of cell lineages would be
present. Several studies have differentiated ESCs in vitro and observed
changes in mitochondrial dynamics during differentiation. As ESCs
differentiate the total number of mitochondria increases, as do the
number of mitochondria with a more mature morphology (St John
et al., 2005a, b; Cho et al., 2006), similar to that described for
SSCs. Concomitantly with an increase of mitochondrial number
during ESC differentiation, the rates of O2consumption and ATP pro-
duction in the cell increase, whereas lactate production decreases
(Chung et al., 2007). These results suggest that during ESC differen-
tiation there is a switch in energy metabolism from glycolysis to
OXPHOS. Similar results have been reported in adult stem cells
(Piccoli et al., 2005; Chen et al., 2008).
It seems logical to assume that an increase in the number of mito-
chondria and OXPHOS in differentiated cells leads to an increase in
ROS production, and several authors have shown that that is indeed
the case (Cho et al., 2006; Saretzki et al., 2008). Interestingly,
reports regarding antioxidant defences during the process of differen-
tiation differ. Although a decrease in the expression of antioxidants,
namely SOD2 and GPX2, has been reported (Saretzki et al., 2008),
increased expression of GPx1, Cu/Zn SOD, Prx1 and Prx2 was
also described (Cho et al., 2006). The contradictory results may be
due to the fact that these studies looked at different antioxidants,
and it is possible that during differentiation there is an increase in
certain antioxidants to the detriment of others.
Mitochondrial role in ESC differentiation
Given the distinct mitochondrial properties in undifferentiated versus
differentiated ESCs it is logical to assume a role for mitochondria
in differentiation. Hypoxic environment prevents spontaneous hESC
differentiation (Ezashi et al., 2005). In addition, several groups have
shown that functional mitochondria are necessary for differentiation.
For example, inhibition of mitochondrial respiratory chain complexes
I and III, by Rotenone and Antimycin A, respectively, results in
reduced cardiomyocyte differentiation, due to an impairment of
OXPHOS (Chung et al., 2007). Furthermore, glycolytic metabolism is
sufficient for maintaining mouse ESC homeostasis; however, in order
for cells to differentiate there must be a switch from glycolysis to the
Figure
(hESCs).(A) Transmission electron microscopy of hESCs with a
large nuclear/cytoplasm ratio and few elliptical mitochondria with
small internal matrix space in perinuclear clusters (asterisks). Bar rep-
resents 2 mm. (B) and (C) Both hESCs (B) and mouse embryonic
feeder fibroblasts (C) were transfected with the Mito-DsRED
vector (red) and imaged by live confocal microscopy. The vector
encodes a fusion red fluorescent protein and the mitochondrial tar-
geting sequence from subunit VIII of cytochrome oxidase. (B) Mito-
chondria distribution in a hESCs undergoing mitosis. Note that the
two daughter cells inherit different numbers of mitochondria. Bar rep-
resents 20 mm. (C) Mouse fibroblast mitochondria are more
elongated that those in hESC. Bar represents 10 mm.
4 Mitochondriainhumanembryonic stem cells
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Page 13

more efficient OXPHOS (Chung et al., 2007). In addition, inhibition of
the complex III of mitochondrial respiratory chain by Antimycin A
reduced the spontaneous appearance of beating foci formed by
differentiating ESCs, probably due to inhibition in calcium signalling
(Spitkovsky et al., 2004). The same inhibitor has been shown to also
boost undifferentiated hESC pluripotency (Varum et al., submitted).
Again, several authors have reported a similar role for mitochondria
in adult stem cell differentiation (Carriere et al., 2004; Chen et al.,
2008). Recently, a correlation between Dc, metabolic rate and the
differentiation of mouse ESCs has been described, with cells with
lower MMP showing more efficient mesodermal differentiation (but
low ability to form teratomas), although a population with higher
membrane potential behaved in the opposite fashion, although both
populations were indistinguishable in terms of pluripotency markers
(Schieke et al., 2008).
Mitochondria and ESC apoptosis
Several authors have reported that mitochondrial apoptotic pathways
play a role in modulating ESC homeostasis and differentiation.
Upon oxidative stress silent mating type information regulation 2
homolog 1 (SIRT1), a deacetylase that catalyzes deacetylation of
acetyllysine residues of proteins such as p53, allows mouse ESCs to
maintain self-renewal by eliminating the cells that were exposed to
endogenous ROS (Vaziri et al., 2001; Han et al., 2008). Under ROS
exposure SIRT1 blocks translocation of the tumor suppressor p53
to the nucleus and induces it’s accumulation in the mitochondria of
mouse ESCs. p53 can then induce mitochondria-mediated cell death
by inducing the release of cytocrome c, SMAC/Diablo and apoptosis
inducing factor (Mihara et al., 2003; Leu et al., 2004; Moll et al., 2006).
Furthermore, p53 suppresses expression of the key regulator of
pluripotency, Nanog, in hESCs (Chambers et al., 2003; Mitsui et al.,
2003; Quin et al., 2007). These elegant studies suggest that SIRT1
maintains mouse ESC self-renewal under stress by inhibition of
p53-mediated suppression of Nanog and by inducing apoptosis of
cells that were exposed to endogenous ROS.
Although not necessarily related to mitochondrial function, it has
been recently reported that caspase 3 mediates both ESC and hema-
topoietic stem cell differentiation (Fujita et al., 2008; Janzen et al.,
2008). Another component of the apoptotic machinery that was
referred as a mediator of stem cell differentiation is Bcl-2, and
mouse ESCs overexpressing this anti-apoptotic protein maintained
pluripotency in serum- and feeder-free conditions (Yamane et al.,
2005). Overall these studies suggest that the mitochondrial apoptotic
machinery, besides it’s canonical role in apoptosis, is an important
mediator of stem cell differentiation.
In summary, although this remains a promising and novel area for
research, overall results indicate that modulation of mitochondrial
activity may be a useful tool to maintain ESCs in a pluripotent state,
or drive differentiation towards a specific lineage. It remains to be
established if the same is true of the more recently characterized
induced pluripotent (iPS) cells, in which a pluripotent ESC-like state
is induced in somatic cells (Takahashi et al., 2007; Yu et al., 2007;
Yamanaka, 2008). Indeed, and although much further research is war-
ranted, both at the basic and applied levels, in all likelihood these iPS
cells will essentially replace current hESC lines in much of the research
related to pluripotency, differentiation, maintenance of a cell state (i.e.
also relevant for putative cell dedifferentiation during cancer),
inasmuch as they also represent a technology with the true potential
for the generation of embryo and oocyte-free patient-specific cell
lines for putative cell replacement therapies.
Conclusions
Mitochondria-based events regulate different aspects of reproductive
function, but these are not uniform throughout the several systems
reviewed. Reversible switches in mitochondrial activity occur through-
out the reproductive system and could reflect changes in substrate
availability or signal profound changes that could be used to modulate
different processes, such as gamete and embryo quality and cell differ-
entiation. Low(er) mitochondrial activity seems a feature of ‘stem-
ness’, being described in spermatogonia, early embryo, inner cell
mass cells and ESCs. Thus, not only do mitochondria mirror, but
also affect cellular state, suggesting the mitochondrial manipulation
affects the differentiation of ESCs (and possibly also iPS cells). Further-
more, recent studies showing unexpected and non-canonical relation-
ships between transcription factors and mitochondrial activity
(Wegrzyn et al., 2009) suggest heretofore unacknowleged levels of
complexity in global cell regulation involving the joint coordination of
signalling, gene expression and metabolism. Future work should
embrace these organelles as targets or indicators of changes
invoked by manipulation, toxic injury and other conditioning factors.
Acknowledgements
All lab members are acknowledged for many fruitful discussions, par-
ticularly Sandra Gamboa (Agricultural School of Coimbra, Portugal).
Anto ´nio Moreno, Paula I. Moreira, Paulo J. Oliveira, M. Sancha
Santos, Teresa Almeida-Santos (University of Coimbra, Portugal),
Bayard Storey (University of Pennsylvania, USA), Christopher Navara
(University of Texas, San Antonio, USA), Gerald Schatten (University
of Pittsburgh, USA), Justin St. John (University of Warwick, UK), Olga
Genbacev and Susan J. Fisher (University of California, San Francisco,
USA) and Stefan Schlatt (University of Muenster, Germany) are also
gratefully thanked for their input on different aspects discussed in this
manuscript.
Funding
S.V., S.A., P.C.M., A.P.S. and A.A. were supported in part by Ph.D.
Fellowships from Fundac ¸a ˜o para a Cie ˆncia e Tecnologia (FCT), Portu-
gal. J.R.-S. was supported by a Fulbright Fellowship and by a Sabbatical
fellowship from FCT, Portugal.
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