Physiological and Pathological Aspects of Sperm Metabolism
Zamira Gibb and Robert John Aitken
Physiological
Aspects of Sperm Metabolism Spermatozoa are highly specialized cells,
playing the vital roles of paternal DNA
delivery and activation of the oocyte
following fertilization. The site of sperm
deposition (in the vagina or uterus in the mammal) is physically removed from the site of
fertilization (the oviduct), and while
a proportion of sperm transport is
facilitated by uterine contractions (in mammals), the spermatozoa must in themselves be
sufficiently motile to traverse the
uterotubal junction and ultimately locate
a single cell, the oocyte. In addition, during
their sojourn within the female tract, spermatozoa must undergo a maturation process called
capacitation in order to attain the
competence to recognize the egg and
then engage in a complex cascade of cell–cell interactions in order to achieve union of the gametes at
fertilization. This process involves extensive
remodelling of the sperm plasma
membrane as well as the induction of
hyperactivated motility and, as such, is a highly energy-dependent process . The
process of spermatogenesis requires extensive
remodelling of a conventional spherical cell to become one of the most highly specialized and
morphologically differentiated cells in
the body. During this transformation, the
DNA in the sperm nucleus reaches the
physical limits of compaction to achieve a quasicrystalline state . This extreme compaction requires the removal or resorption of most of the
cytoplasm, at the same time removing
the majority of the organelles (such as
the endoplasmic reticulum, ribosomes and
Golgi apparatus) that are intimately involved in the regulation of metabolism in somatic cells.
The result of this extensive
remodelling is that spermatozoa are left
translationally and transcriptionally silent, as well as relatively depleted of intracellular
enzymes and energy reserves such as fat
droplets, yolk granules and glycogen . For this reason, spermatozoa are heavily dependent on their immediate extracellular
environment for the energy substrates
that drive metabolism, as well as a
variety of specialized enzymatic activities
that would normally be conducted
intracellularly . For example, in somatic cells, the array of
enzymes and low-molecular-mass
scavengers involved in mediating protection
against oxidative stress is housed intracellularly, largely within the cytoplasmic space.
Spermatozoa, on the other hand, largely
depend upon the epididymal and seminal
plasmas to provide the richest and most
diverse combination of antioxidants in the
body, including several that are unique to the male reproductive tract , . In much the same way that economies trade
using a currency rather than a barter
system, biological systems have all
evolved their own unique ‘currencies’ for
the exchange of energy. The most important of these currencies is adenosine ’-triphosphate (ATP), which provides the metabolic energy to drive
activities in all living cells. Cellular Respiration The generation of ATP may be achieved either
in the presence (aerobic) or in the
absence (anaerobic) of oxygen. At the
advent of life on Earth, the atmosphere
was almost if not entirely devoid of oxygen, and at this time anaerobic glycolysis presented the only
metabolic pathway by which organic
molecules might be broken down with the
release of energy. However, as glycolysis
typically requires large quantities of carbohydrates such as sugars, which would not have been
available to the earliest life forms,
it has been suggested that The Sperm
Cell, Second Edition, ed. Christopher J. De Jonge and Christopher L. R.
Barratt. Published by Cambridge University
Press. [1]C Cambridge
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Sperm Metabolism during this period of
substrate famine, the phosphorylation of
adenosine diphosphate (ADP) to produce ATP
was driven by the energy released during the
cleavage of the carbon–nitrogen bonds of amino acids such as glycine . Following the emergence of eukaryotic plant
cells as a result of the association
between glycolytic and photosynthetic
prokaryotes, the levels of atmospheric oxygen
began to increase, giving rise to oxidative
metabolism and the endosymbiosis of the mitochondrion to form the first animal cells. The ability
to exploit the highly reactive oxygen
molecule as the driving force behind
ATP production results in an extremely
effective pathway for energy generation
which, while significantly more efficient than utilizing glucose, produces significant quantities of
reactive oxygen species (ROS) as a
by-product . As with somatic cells, the predominant
metabolic pathways that spermatozoa use
to produce ATP are glycolysis and
oxidative phosphorylation (OXPHOS), with
the preferential pathway utilized by spermatozoa of various species depending on a number of
factors, including oxygen and hexose
availability .The enzymes necessary for glycolysis are
primarily associated with the fibrous
sheath located in the principal piece
of the tail .
In contrast, OXPHOS occurs in the
mitochondrial gyres located in the midpiece.
OXPHOS is a significantly more efficient method of ATP production than glycolysis. Despite
this, spermatozoa from most heavily
researched species, including humans
and laboratory rodents, depend predominantly
on glycolysis for ATP production . During
spermatogenesis, DNA is condensed to a crystalline structure which not only provides mechanical
protection from ROS damage, but also
allows the spermatozoa to become
streamlined for ease of movement . During this process, the majority of the
cytoplasm is removed from the cell, and
as a result, spermatozoa have limited
intracellular space to store energy reserves
in the form of glycogen, lipid droplets or yolk granules and are almost entirely dependent
on external substrates such as fructose
for glycolysis and lactate, citrate or
succinate for OXPHOS. Glycolysis The role of glycolysis in driving the production
of ATP for motility has been well
researched due to its relative importance
in humans and laboratory species. Large
polar molecules such as glucose cannot diffuse
across membranes, and their transport is facilitated by membrane-bound proteins, which were first described by Kasahara and Hinkle in 0 .These proteins may be broadly divided into two
groups, the ATP-dependent
sodium-coupled glucose transporters (SGLTs)
and the facilitative glucose transporters
(GLUTs),which allow passive transport of sugars across the membrane 0, .
Of these, GLUTs are significantly more
abundant and have received a great deal
more attention than SGLTs. GLUTs are categorized according to their relative ability to
transport hexoses (such as glucose,
mannitol and fructose), amino sugars
and vitamins .
Since the discovery of the glucose
transporter GLUT , a great many
additional GLUTs have been
characterized , .While GLUTs , ,
and appear
to be the most abundant GLUTs expressed
by spermatozoa (Table . ) ,
, GLUTs , a
and b have also been described ,
0
. The pattern of GLUT distribution,
which is largely confined to the acrosome and principal piece of the sperm cell, suggests
that glycolytic processes are involved
in generating energy for the membrane
modifications required for hyperactivation
and the acrosome reaction. Should this be the case, the distribution of GLUTs would be
expected to change with the functional
status of the cell (i.e. between
noncapacitated and capacitated states), a phenomenon which has been reported in the dog, but has not been observed in other species . At this
stage, the significance of active glycolytic pathways in OXPHOS-dependent spermatozoa (e.g.
equine) for the production of ATP for
either motility or capacitation and the
acrosome reaction remains poorly understood. Despite the fact that spermatozoa are able
to take up sugars and utilize them as
energy sources, the extracellular
glycolytic substrate availability in vivo is
scarce. The concentration of glucose and other reducing hexoses in epididymal fluid is in trace or
nondetectable amounts ,
,
and in the oviduct is generally in
micromolar concentrations .
However, in the dog at least, the
ability of spermatozoa to engage in
gluconeogenesis by utilizing stored glycogen
may be sufficient to provide the glucose necessary for glycolysis ,
. Interestingly, in the presence of a sufficient concentration of glucose,
dog spermatozoa actively store glycogen
,
providing a buffer against periods of
hexose starvation. Seminal plasma contains
a relative abundance of fructose and glucose
,
and the ability to store energy efficiently Physical distribution of glucose
transporters in mammalian spermatozoa during
the brief exposure that spermatozoa have to
this nutrient-rich fluid following ejaculation may provide the energy required to ascend the female
reproductive tract and fertilize the
oocyte. Although glycogen, glycogen
synthetase and glycogen phosphorylase have
also been described in spermatozoa of the ram,
boar and horse ,
gluconeogenesis mechanisms are yet to
be demonstrated in any species other than the
dog. However, the ubiquitous distribution of GLUT and the presence of enzymes associated with
gluconeogenesis and glycogen synthesis
suggest that the utilization of stored
glycogen is likely to play a role, at least in
the mammal, in the maintenance of energy homeostasis and sperm survival in vivo. Although mitochondrial inhibition studies
have highlighted the contribution of
OXPHOS to energy production by
spermatozoa ,
deciphering the relative importance of
glycolysis to sperm function is somewhat
more problematic. Many studies have attempted
to quantify the relative contribution of glycolysis by inhibiting this process using the
biologically unavailable isomer -deoxy-D-glucose. While this method does indeed result in a decrease
in ATP, it is a somewhat blunt
instrument in that the depletion of ATP
is due to both the absence of usable hexoses and ATP exhaustion through the futile
phosphorylation of the unusable -deoxy-D-glucose by hexokinases a highly
energy-dependent activity .
A more direct https:/www.cambridge.org/core/terms.
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: Physiological and Pathological
Aspects of Sperm Metabolism approach to
the quantification of glycolytic processes
would be to perform flux and product distribution measurements using C-labelled glucose and evaluating the production of C-labelled pyruvate and lactate in the presence and absence of
mitochondrial inhibition.
Oxidative
Phosphorylation
An excellent
example of a species with OXPHOS dependent
spermatozoa is the horse. Despite the well characterized presence of GLUTs on stallion sperm, it has become abundantly evident that these
spermatozoa differ from those of other
well-studied mammalian species, in that
their energy demands are met not by glycolytic
pathways, but by using OXPHOS , ,
0 ; in the presence of
mitochondrial inhibitors, they suffer a
rapid loss of velocity and a dramatic decline in ATP content, while glycolytic human spermatozoa
display no significant decrease in
motility parameters or ATP levels . This dependence results in an
unconventional positive relationship
between reactive oxygen species (ROS)
production and fertility in the stallion
, , 0 ,
with the source of ROS being the mitochondrial
electron transport chain, within which about
% of
O reduced in the mitochondria during OXPHOS forms superoxide . While
human clinical data consistently report negative correlations between male fertility and
sperm oxidative stress ,
, a recent study has revealed a paradoxical inverse relationship between
fertility and the percentage of live
cells without oxidative damage in the
OXPHOS-dependent spermatozoa of the stallion
.
In addition, spermatozoa from matings which
resulted in a conception (therefore considered
to be more fertile) had lower vitality and a higher percentage of cells displaying ROS-induced damage than spermatozoa from matings which did not
result in a conception upon arrival at
the laboratory. During in vitro storage
and transport of the samples, the more metabolically
active spermatozoa from the more fertile
stallions were becoming exhausted at a higher rate, so that by the time that the assays were
performed in the laboratory, the cells
had suffered an accelerated demise due
to the accumulation of metabolic byproducts,
such as ROS and cytotoxic lipid aldehydes. Essentially, OXPHOS-dependent spermatozoa ‘live fast and die young’.
To avoid the introduction of artefacts following glycolytic inhibition ,
a relatively simple method of
elucidating the mode of ATP generation by spermatozoa may be achieved through the inhibition of OXPHOS using mitochondrial uncouplers such
as carbonyl cyanide m-chlorophenyl
hydrazone, Antimycin A, rotenone or
diphenylene iodonium. In the stallion, OXPHOS
inhibition results in over a 0%
reduction in sperm velocity and a % reduction in sperm ATP levels, while human sperm velocity and ATP remain
unaffected. In addition, the greater
efficiency of OXPHOS mediated ATP
production supports a higher velocity, and
indeed stallion sperm velocity parameters are
around 0% faster than those of
glycolysis-dependent human spermatozoa . Ultimately,
high ROS production by OXPHOS dependent
spermatozoa appears to be a physiologically normal scenario brought about by superoxide leakage from the mitochondrial electron
transport chain during OXPHOS , with a positive relationship between mitochondrial ROS production and sperm velocity, leading to increased rates
of lipid peroxidation and,
following prolonged storage, a loss of
motility and vitality .
This phenomenon has a number of
implications for the in vitro storage of
OXPHOS-dependent spermatozoa, since the prolonged generation of ROS in the absence of
extracellular free radical and lipid
aldehyde scavengers will lead to
irreversible oxidative damage, impairing DNA
integrity and sperm functionality.
The Translocation of ATP around the
Sperm Cell In the majority of
somatic cells, the mitochondria are located
within the cytoplasm and are therefore able
to deliver ATP from the site of production to the sites of utilization in an effective and
efficient manner. This system is rather
more problematic for cells such as
spermatozoa, in which compartmentalization
has resulted in mitochondria being physically disconnected from the fibrous sheath of the tail where
ATP is most heavily required for
motility. This anatomical anomaly has
led to an unwillingness on the part of
many researchers to acknowledge that for many
species mitochondrial ATP production plays a vital role in the energy homeostasis of
spermatozoa. While several plausible
simple ATP diffusion models have been
postulated ,
these models do not account for the
need to remove and recycle ATPase byproducts
such as ADP, Pi and H+. https:/www.cambridge.org/core/terms.
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Sperm Metabolism It has been suggested
that in spermatozoa the movement of ATP
and ADP between the sites of production
(the mitochondria and the cytoplasm) and the sites of utilization occurs through flux
transfer chains .These depend upon the rapid transfer of the
displacement from equilibrium of an
enzyme reaction down a chain of
adjacent enzymes, such that an ATP added
at one end could effectively be removed at the
other end. Such enzymatic shuttle systems not only facilitate the delivery of ATP to the sites
of utilization, but also remove and
recycle ATPase byproducts . This equilibrium displacement process may
proceed with either creatine kinase or
glycolytic reactions, and is far more
rapid and efficient than the diffusion of
reactants ,
a feature of particular importance to species
with extremely long sperm flagella, such as the rat . Modulation
of Metabolism: In vitro Storage of
Spermatozoa In vitro sperm storage is
often necessary for a number of reasons
associated with assisted reproductive technologies
such as artificial insemination (AI) and
in vitro fertilization (IVF). During the final
phases of spermatogenesis, spermatozoa lose the ability to biosynthesize, repair, grow and divide,
becoming remarkably simple in their
metabolic functions . Typically, sperm ageing and the inevitable
senescence that follows can be delayed
or even arrested through the
implementation of temperature-induced metabolic
restriction by chilling or cryopreservation.
The phenomenon of cold-induced sperm preservation was first discovered by Spallanzani in . He observed that cooling of frog, stallion
and human semen in snow did not kill
all the ‘spermatic vermicules’, but
rendered them temporarily immotile and induced
a state of lethargy from which they could
recover when returned to higher temperatures. By restricting the metabolic rate of cells, the
production of toxic metabolic
byproducts, such as hydrogen peroxide,
lipid aldehydes and carbon dioxide, is reduced
and the depletion of ATP associated with
the maintenance of homeostasis , , is minimized.
This temperature-induced metabolic restriction reduces the rates of both ROS production and acidification of the storage medium through
the accumulation of lactic acid and CO from
glycolysis and OXPHOS, respectively.
However, the spermatozoa of many
stallions, human male patients and other species do not tolerate the stresses associated with
chilling or cryopreservation
particularly well .Therefore, there has recently been a concerted effort
to develop media that will extend the
longevity of spermatozoa without the
need to chill or cryopreserve . The major advantage of chilling semen is a
reduction in sperm metabolic rate that
results in improved longevity during
transport and storage and limits the
growth of harmful bacteria. Temperature-induced reduction of sperm metabolism is of
particular importance in the case of
OXPHOS-dependent spermatozoa . If metabolism is not curtailed in these
cells by temperature reduction, OXPHOS
will produce significant quantities of
ROS ,
which invariably compromise sperm
function , .
Second, depletion of ATP is known to
compromise a wide range of ATP dependent
functions in spermatozoa that are necessary to maintain homeostasis and prevent
premature cell death .Therefore, it is clear that in an ambient temperature storage medium, mitochondrial energy production must be supported while
unnecessary ATP depletion is minimized,
as a result of pressure placed on
ATP–dependent pathways such as the regulation of ionic or osmotic flux ,
. Supplementation
of ambient-temperature semen extenders
with various antioxidants , ,
nutrient substrates and osmolytes has
gone a long way toward ameliorating the
detrimental effects of ROS production
and ATP depletion in spermatozoa during
storage at ambient temperatures. A temperature-independent
mechanism for inhibiting sperm
metabolism would provide the ideal solution
for ART systems, avoiding the irreversible membrane damage induced by cooling and freezing,
while avoiding the accumulation of
toxic metabolic byproducts and the
depletion of ATP. The clue to such a strategy
may be found by taking a closer look at the in vivo environments that are conducive to sperm
longevity, namely the epididymis and
the oviductal isthmus, where
spermatozoa are stored in a quiescent state for considerable periods of time. Pathological Aspects of Sperm Metabolism
In parallel with recent advances in our understanding of the metabolic pathways that drive normal
sperm function, we have seen
significant advances in our understanding
of the metabolic mechanisms responsible
for defective sperm function. In this context there https:/www.cambridge.org/core/terms.
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Sperm Metabolism is a general consensus
that one of the major mechanisms responsible
for loss of sperm motility, fertilizing
potential and DNA integrity is oxidative stress. In many ways ROS are a two-edged sword when it
comes to the regulation of normal sperm
function. They are required in small
quantities to promote cellular processes
such as capacitation ,
but when generated in excess, they can
become extremely damaging because of
their promiscuous reactivity. In the remainder of this chapter we shall examine both aspects of
these biologically important molecules
in the context of sperm cell biology. However,
in order to set the scene, we shall first
overview the fundamental chemistry of ROS and
consider the difficulties encountered in detecting these short-lived but highly reactive molecules. What Are Reactive Oxygen Species? The term ‘reactive oxygen species’ covers a
range of metabolites that are derived
from the reduction of oxygen, including
free radicals, such as the superoxide anion
(O − •) or the hydroxyl radical (OH•),
as well as powerful oxidants such as
hydrogen peroxide (H O ). The
term also covers molecular species derived from the reaction of carbon-centred radicals with
molecular oxygen, including peroxyl
radicals (ROO•), alkoxyl radicals (RO•)
and organic hydroperoxides (ROOH). The
term ‘ROS’ may also refer to other powerful oxidants such as peroxynitrite (ONOO-) or
hypochlorous acid (HOCl) as well as the
highly biologically active nitrogen
free radical nitric oxide (•NO). The
specific term ‘free radical’ refers to any atom or molecule containing one or more unpaired
electrons. As unpaired electrons are
highly energetic, and seek out other
electrons with which to pair, they confer
considerable reactivity upon such radicals. Thus, free radicals and related ‘reactive species’ have
the ability to react with, and modify,
the structure of many different kinds
of biomolecules including proteins, lipids
and nucleic acids. The wide range of targets that can be attacked by ROS is a critical facet of
their chemistry that contributes
significantly to the pathological importance
of these molecules. As most chemical species
in biological systems have only paired electrons, free radicals are also likely to be involved
in chain reactions, whereby new free
radical products are formed. A classic
example of such a chain reaction is the
peroxidation of lipids in biological membranes. In this process, a ROS-mediated attack on
unsaturated fatty acids in the plasma
membrane generates peroxyl (ROO•) and
alkoxyl (RO•) radicals that, in order to
stabilize, abstract a hydrogen atom from an adjacent carbon, generating the corresponding acid
(ROOH) or alcohol (ROH). The
abstraction of a hydrogen atom from an
adjacent lipid creates a carbon-centred radical that combines with molecular oxygen to
create another lipid peroxide. In order
to stabilize, the latter must abstract
a hydrogen atom from a nearby lipid, creating
yet another carbon radical that on reaction with molecular oxygen will generate more lipid
peroxides. In this manner, a chain
reaction is created that propagates the
peroxidative damage throughout the plasma
membrane. Since the hydrogen
abstraction process referred to above
is facilitated by the double bonds present in
unsaturated fatty acids, membranes that are rich in the latter will be particularly vulnerable to
oxidative stress. In this context,
spermatozoa are especially susceptible because
their plasma membranes are extremely rich
in unsaturated fatty acids,
notably : 0 . Such an
abundance of unsaturated lipids is necessary to create the membrane fluidity required by the
membrane fusion events associated with
fertilization (acrosomal exocytosis and
sperm–oocyte fusion); however, their presence
leaves these cells open to peroxidative attack. Termination of such lipid peroxidation chain
reactions can be achieved with
chain-breaking antioxidants such as
vitamin E (-tocopherol). The latter is extremely effective in terminating lipid peroxidation
cascades in human spermatozoa in vitro and
has also been shown to improve the
fertility of males selected on the basis
of high levels of lipid peroxidation in their spermatozoa, in vivo . The most commonly encountered ROS are O − • and
H O
. These molecules are capable of a range of rapid chemical reactions yielding a
correspondingly broad range of reaction
products. When in aqueous solution, O − • has a short half-life ( ms) and is
relatively inert. The radical is more stable and reactive in the hydrophobic environment provided by
cellular membranes. The charge
associated with O − • means that this molecule is generally
incapable of passing across biological membranes,
although there are reports of this
molecule passing through voltage dependent
anion channels .As
a result of its lack of membrane
permeability, O − • may be more
damaging if produced inside biological membranes
than at other sites. It is also
important to note that whileO −• can
act as either a reducing agent or a
weak oxidizing agent in aqueous
solution, under the reducing conditions that https:/www.cambridge.org/core/terms.
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Sperm Metabolism characterize the
intracellular environment, O − • acts primarily as an oxidant. Many of the effects
of O − • are believed to arise from its conversion to
more reactive oxidizing species , .
For example, protonation of O − • forms the hydroperoxyl radical (HO •), a much
stronger oxidant: HO • [1] H+ + O − •. The
pH at which this reaction reaches equilibrium
(pKa) is . , with the result that at physiological pH, HO •
represents less than % of the O − • present in a cell. However, given the considerable
reactivity and membrane permeability of
HO •, this radical is still believed to be a significant contributor to
oxidative damage in biological systems,
with the potential to initiate lipid
peroxidation cascades , . Conversion of O − • to other ROS also occurs. An important means by which this takes place is
the dismutation reaction, wherein O − • reacts with itself (i.e. superoxide is both oxidized and reduced). In
this situation, one molecule of O − • is oxidized to molecular oxygen, while the other is reduced to H O : O −
• + O − • + H+ → H
O + O . Superoxide
dismutase (SOD) catalyzes this conversion.
SODs are metalloenzymes thought to be present in all oxygen-metabolizing cells .The reaction can occur spontaneously without SOD; however, in
its absence, dismutation will proceed
much more slowly due to the
electrostatic repulsion of the anions (rate
constant of about × 0 M− s− at physiological pH). SOD is an efficient catalyst that will drive
the above reaction at a rate constant
of about . × 0 M− s−
over a wide pH range ( . . ).
In the human spermatozoon, there is
sufficient SOD activity to account for all
of the H O produced
by these cells . Superoxide formation can also lead to the
generation of other types of highly
reactive species apart from H O .
Many transition metal ions are able to participate in these processes, as they possess variable
oxidation numbers, permitting them to
change their redox status by either
gaining or losing an electron. Consequently,
transition metals act as very effective promoters of free radical reactions. For example, in
the Fenton reaction, H O undergoes decomposition in the presence of ferrous ions to produce the pernicious
hydroxyl radical (OH•): H O + Fe + → Fe
+ + OH− + OH•, Fe + + O
− • → Fe + + O . The
sum of these two reactions represents the iron catalyzed Haber–Weiss reaction: H O + O − • → O
+ OH− + OH•. Thus,
O − • also has a key role to play in
the above reaction by serving as a
reductant and facilitating the regeneration
of reduced metal ions in the extracellular
space. It is also well recognized that other transition metals may participate in these reactions.
Thus, while iron is the major player , copper is the other major candidate and cobalt, aluminium,
chromium, nickel and titanium may also
participate in such reactions 0 .Moreover, in seminal plasma, both iron and copper are available in a free state and
hence able to take part in OH•
production and the consequent promotion
of oxidative stress in the ejaculate . Detection
of ROS in the Male Germ Line Given the
importance of ROS and the apparent vulnerability of spermatozoa to oxidative stress, it might be anticipated that sophisticated methods
would have been developed to detect
these intermediate oxygen metabolites
for diagnostic purposes. In fact, this area
has been severely compromised by the absence of sensitive, accurate analytical methods capable of
confirming the presence of specific ROS
in biological systems. The most
commonly used method for detecting ROS
in an andrological context is chemiluminescence, using the probe lucigenin or luminol , . Lucigenin (N,N’-dimethyl- , ’-biacridiniumdinitrate) carries a positive ionic charge; it is
generally thought to be relatively membrane-impermeant
and to respond to O − • in the extracellular space. However, the positive charge associated with this
molecule may also favour its partition
into mitochondria, as a consequence of
the electronegative mitochondrial membrane
potential. Indeed, studies using rat spermatozoa as a model indicate that the lucigenin
signal generated by these cells can
reflect O − • produced by the sperm mitochondria . However, there are no data to suggest that the lucigenin signals generated by
human spermatozoa are of mitochondrial
origin, even though such signals are
inversely correlated with sperm quality
and significantly elevated in cases of male
infertility ,
. One
of the key features of lucigenin is that this
probe must undergo a one-electron reduction to the radical species, LH+•, before it becomes
sensitized to the presence of O − • (Figure . ).
In the case of https:/www.cambridge.org/core/terms.
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Sperm Metabolism NAD(P)H Cellular O -•
LucH+• Dioxetane formation Chemiluminescence LucH+•
+ = + + e- - e- + LucH+• LucH O
_• O O
-• HO
- SOD Lucigenin reduction e.g., Cytochrome P 0
reductase Cytochrome b reductase
H O Luc
+ LucH+• Luc + Figure
.
Schematic representation of the
chemistry for lucigenin chemiluminescence; Luc
+: lucigenin; LH+•: a lucigenin radical created by the one–electron reduction of Luc +. The reaction of LH+• with oxygen
generates O − •. The latter then
participates in an oxygenation reaction
with LH+•, generating a dioxetane that decomposes with the generation of
chemiluminescence. Any entity that can effect the one-electron reduction of lucigenin can
potentially create a redox cycle in the presence of oxygen that produces high
levels of O − • and chemiluminescence. It is impossible to
distinguish the relative contribution of such probe-dependent and
cell-dependent chemiluminescence. Hence
data obtained with this probe should be interpreted with caution. mitochondrialO − • production, this reductive process is accomplished by the organelle’s electron
transport chain. However, outside of
the mitochondria, lucigenin reduction
can be induced by reductases such as cytochrome
P 0 reductase or cytochrome b reductase
.This is particularly the case when
exogenous NAD(P)H is used to drive
redox activity in populations of human
spermatozoa .The
LH+• generated on reduction then
combines with O − • to produce the dioxetane that, in turn, decomposes with the
generation of light
(chemiluminescence): Although this
chemistry seems straightforward, complications
may arise due to redox cycling reactions
whereby LH+• combines with ground state oxygen (O )
to create O − • and regenerate the
parent lucigenin molecule (Figure . ).The
O − • artificially created in this manner will then combine with LH+•
to generate additional dioxetane and
further the chemiluminescence response
(Figure . ). If such redox cycling does occur, the particularly intense
NADPHdependent lucigenin signals seen
in defective human spermatozoa may be
as much an indication of excessive reductase
activity as evidence for the overabundance
of O − • .This explanation would provide a link between the high levels of redox
activity detected in defective
spermatozoa by lucigenin chemiluminescence
and enhanced reductase activity due to the presence of excess residual cytoplasm. There is
certainly a great deal of data to link
cytoplasmic retention with defective
sperm function 0 ,
so such an explanation would be fully
compatible with our understanding of
the etiology of male infertility. It
has also been argued that the reaction of LH+•
with O is thermodynamically unlikely and
that redox cycling of this probe does
not occur in biological systems.
However, given the high dose of lucigenin
typically used to detect redox activity in human sperm samples ([1] 0 μM), the possibility of spurious
results generated by continuous cycling
of the probe cannot be excluded. As a
consequence, we currently do not know
the extent to which the elevated lucigenin signals detected in defective human spermatozoa
reflect primary O − • production or the superabundance of reductases due to the presence of excess
cytoplasm. What we do know is that the
activity of this probe correlates well
with defective sperm function whether the
activity is promoted by treatment with NAD(P)H or phorbol ester ,
, ,
. A
similar argument may apply to luminol. This
probe has to undergo a one-electron oxidation before it becomes sensitized to the presence of
ROS. In a common form of this assay,
horseradish peroxidase is used to
promote luminol oxidation in the extracellular
space. In this form, the luminol assay largely reflects the presence ofH O released to the outside of the cell. In the absence of exogenous horseradish
peroxidase, https:/www.cambridge.org/core/terms.
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, subject to the Cambridge Core terms of use, available at Chapter : Physiological and Pathological Aspects of
Sperm Metabolism e.g., Cytochrome P 0
reductase LucH+. LucH+. LucH Luc +. LucH+. O H O HO
− O −.
SOD Luc +. + e− − e− LucH+.
O −. Dioxetane formation Chemiluminescence + + = + Lucigenin
reduction NAD(P)H Cellular O
−. Cytochrome b reductase
Figure . Schematic representation of the underlying
chemistry for luminol-dependent chemiluminescence. L: luminol; L•: a luminol radical created by the one-electron
oxidation of L; L+: azaquinone formed by the further one-electron oxidation of
L• by oxygen, generating O − • as a byproduct. The reaction of L• with
O − • or L+ with H O generates an unstable endoperoxide whose
decomposition leads to production of
the chemiluminescent species, an electronically excited aminophthalate. Redox
cycling of the probe could result if human
spermatozoa possessed an appropriate reductase to convert L+ back to the
parent L. Any reactant that can achieve the univalent oxidation of luminol will generate chemiluminescence in
this assay, including H O and
ONOO_. the assay is dependent on the
presence of intracellular peroxidases
to activate the probe .
The oneelectron oxidation of luminol
leads to the creation of a radical
species (L•). The latter then interacts with
ground state oxygen to produce O
− •, which induces the
oxygenation of L• to create an unstable endoperoxide, which ultimately breaks down with the
release of light (Figure . ).
According to this scheme, O − • is an essential intermediate in the creation
of luminol dependent chemiluminescence
and it is for this reason that SOD is
such an effective inhibitor of this reaction
cascade.However, the activity of this scavenger should never be taken to indicate the primary
production of O − • by human spermatozoa; O − • is simply an artificially created intermediate that is essential for
luminol dependent chemiluminescence.
Indeed, any univalent oxidant has the
potential to generate O − •, and hence chemiluminescence, in the presence of
luminol, including ferricyanide,
persulphate, hypochlorite, ONOO and xanthine oxidase (Figure . ).
Hydrogen peroxide lies upstream of O − • in the reaction scheme depicted in Figure . and its involvement in the initial oxidation of luminol accounts for the
inhibitory effects of catalase on this
form of chemiluminescence. In addition,
H O
will also react directly with
azaquinone (L+) and thereby contribute
to the formation of excited
aminophthalic acid, the chemiluminescent
species . Fundamentally,
luminol-based assays are measuring redox
activity characterized by the cellular generation
of oxidizing species capable of creating
L•. Notwithstanding the reservations that might be expressed concerning the specificity of
this probe, the luminol assay is robust
and generates results that are strongly
correlated with sperm function , . The clinical significance of this assay has
also been emphasized in a long-term
prospective study of couples
characterized by a lack of detectable pathology in the female partners. In this cohort of
patients, a negative association was
observed between luminol dependent chemiluminescence
and the incidence of spontaneous
pregnancy .
Furthermore, within this data set, the
conventional criteria of semen quality were
of no diagnostic value whatsoever . A recent detailed analysis of these
chemiluminescent probes confirmed that
lucigenin is not a particularly useful
probe for measuring ROS generation by
human spermatozoa 0 .
Furthermore, while the combination of
luminol and peroxidase was found to be
a sensitive means of detecting ROS in
Use of DHE as a flow cytometry probe for detecting the
generation of O − • by populations of
spermatozoa. A, this probe can be non-specifically
oxidized to generate the parent ethidium (Eth); however, reaction with O − • generates a ROS–specific DNA-sensitive fluorochrome, -hydroxyethidium ( OH Eth). B, hplc analysis of the
fluorochromes generated by human spermatozoa has demonstrated the presence of both Eth and -OH Eth, confirming the generation of O − • by these cells. C, the combination of
DHE and Sytox green (green or yellow
where cells are nonviable) provides an extremely efficient means of
demonstrating ROS generation by viable cells (nuclei stained red with no trace of yellow or green staining).
(A black and white version of this figure will appear in some formats. For the
colour version, please refer to the
plate section.) extracellular space, it
was also found to be very susceptible
to interference by free-radical-generating
leukocytes 0 .
Differentiation of the luminol signal generated
by human sperm suspensions into the component
generated by leukocyte contamination and the component generated by defective spermatozoa
still confounds the interpretation of
studies employing this probe. The problem of cellular contamination has
been solved by the use of flow
cytometry to focus on the cellular
generation of ROS by spermatozoa while gating
out any contribution made by other cell types.The most commonly used flow cytometry probes are
dihydroethidium and mitoSox red (MSR).
These probes are both reduction
products of ethidium bromide, but MSR
has been chemically modified to give the
molecule a positive charge that results in its concentration in the mitochondrial matrix. When run in conjunction with a vitality stain such as
Sytox green (Figure . ),
these probes provide a sensitive, effective
and accurate means of assessing ROS generation by spermatozoa 0 . A potential problem with DHE and MSR is that they can be
non-specifically oxidized to generate
the parent ethidium molecule and a
positive signal in the assay. In order to be certain that the activity probes are reflecting
intracellular ROS generation, it is
important to establish that - hydroxyethidiumis being generated in the
presence of spermatozoa, since this
reaction product is only produced when
these probes are oxidized by O − •.
Fractionation of the fluorochromes
generated by human spermatozoa, followed
by mass spectroscopy, has https:/www.cambridge.org/core/terms.
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, subject to the Cambridge Core terms of use, available at Chapter : Physiological and Pathological Aspects of
Sperm Metabolism 0 0
0 0 0
00 0 0 . . . y = −
. x + 0.
, r = 0 . 0 MDA +
HA (μmol) A hour incubation in vitro 0
0 0 0
0 00 0
0 . . . . . .
MDA + HA (μmol) y = −
. x + 0.0 , r = 0. B Xanthine oxidase system Figure . Sperm motility is negatively impacted by lipid
peroxidation. Using an assay that records the generation of malondialdehyde and -hydroxyalkenals (MDA+ HA), it is possible to demonstrate a clear
negative correlation between sperm motility and A, the generation of lipid peroxides by spermatozoa following an
overnight incubation at °C and B,
the enzymatic creation of oxidative stress using xanthine oxidase . confirmed
the generation of -hydroxyethidium and thus O
− • by these cells (Figure . ).With
MSR as a probe to assess the ability of
human spermmitochondria to generateO − •, electron leakage frombothcomplex I and complex III has been demonstrated . When
the generation ofO − • occurs on
thematrix side of the
innermitochondrialmembrane at complex I, the
result is the induction of lipid peroxidation and motility loss, whereas ROS generation at complex III
leads to the rapid formation of
hydrogen peroxide, which rapidly exits
the cell and enters the extracellular space
.
MSR and DHE are excellent probes for mitochondrial andtotal cellular ROS respectively,
responding readily to stimulants of ROS
generation such as lipid aldehydes,
menadione and catechol oestrogens 0 . Recently, novel boronate probes have been reported for human spermatozoa that are more
sensitive for the detection of ROS than
DHE, MSR and ’, ’-dichlorohydrofluorescein
diacetate (DCFH). These reagents may
well have clinical utility for the diagnosis
of oxidative stress inthemale germline . Impact
of Oxidative Stress on Spermatozoa The
clinical significance of oxidative stress in the etiology of defective sperm function was first
indicated by Thaddeus Mann and
colleagues at the University of Cambridge,
years ago 0 .These authors observed a correlation between the lipid peroxide
content of human spermatozoa and
severemotility loss.This relationship between
motility loss and oxidative stress is
striking and has been repeatedly demonstrated in independent studies .Thus exposure of human spermatozoa to extracellularly
generated ROS induces a loss of
motility that is directly correlated
with the level of lipid peroxidation experienced by the spermatozoa (Figure . ).
Similarly, the loss of motility
observed when spermatozoa are subjected
to overnight incubation is highly correlated
with the lipid peroxidation status of the spermatozoa at the end of the incubation period (Figure . ). The prognostic value of stress tests based
on the loss of motility observed when
spermatozoa are incubated for defined
periods of time in the presence of transition
metals is probably another reflection of the importance of lipid peroxidation in the
modulation of sperm function. The
ability of antioxidants to preserve sperm
motility in vivo and in vitro is yet more evidence that lipid peroxidation is a major cause of motility loss in populations of human spermatozoa . Recently,
studies have been conducted using animal
models exhibiting infertility associated with oxidative stress, such as the GPx knock-out mouse and testicular heating, that have clearly demonstrated the
therapeutic potential of antioxidants
in vivo Physiological and Pathological Aspects of Sperm Metabolism The mechanisms by which lipid peroxidation
leads to motility loss probably involve
changes in the fluidity and integrity
of the plasma membrane and a subsequent
failure to maintain membrane functions critical
to flagellar movement. Disruption of membrane
Ca +/Mg + ATPase activity as a consequence of decreased membrane fluidity would, for
example, lead to motility loss
secondary to an increase in intracellular
calcium. In addition, the electrophilic lipid aldehydes generated as a consequence of lipid
peroxidation are known to form adducts
with the nucleophilic centres of
proteins involved in the orchestration of sperm movement, such as the dynein heavy chain 00 . Of
course, lipid peroxidation will also disrupt all sperm functions dependent on membrane activity,
including sperm–oocyte fusion and the
ability to undergo a physiological
acrosome reaction . Oxidative stress is also amajor cause of DNA
damage in human spermatozoa. Using
quantitative PCR to calculate lesion
frequency, the mitochondrial genome has
been shown to be much more susceptible to DNA
damage than the nuclear genome 0 . As a consequence, the integrity of the sperm mitochondrial genome is an excellent marker of oxidative
stress, even though this genome is of
no biological significance in its own
right because sperm mitochondria do not
generally replicate after fertilization. When quantitative PCR was used to compare the lesion
frequencies induced in spermatozoa and
a variety of other cell types following
exposure toH O , the nuclear genome of the male gamete was shown to be
particularly resistant to oxidative
damage. This resistance is thought to mirror
the unique manner in which nuclear chromatin
is packaged in spermatozoa, as reflected in the high levels of irradiation required to damage
sperm DNA compared with somatic cells 0 . Adequate compaction and stabilization of
sperm nuclear DNA is therefore critical
for protecting this material from
oxidative stress. Amongst Eutheria, spermatozoa
of human origin appear to be more susceptible
to DNA damage than those of most other
species. This is largely because the P
protamine, characteristic of human spermatozoa, has a limited number of thiol groups for disulphide bonding 0 .
Furthermore, the protamination of human
spermatozoa is notably inefficient,
with around % of the genome remaining histone-rich, even in normal
fertile men 0 .
Failures in either the ability of the testes to adequately protaminate human sperm chromatin or the ability of the epididymis to support
subsequent protamine cross-linkage lead
to imperfections in the state of
chromatin stabilization. Such deficiencies in chromatin packaging have, in turn, been associated
with an increased risk of DNA damage 0 .
The particular vulnerability of poorly
compacted sperm DNA to oxidative attack
results in high levels of the oxidative
base adduct, -hydroxy- ’-deoxyguanosine ( OHdG),
being present in human spermatozoa 0 , 0 .
The spermatozoon has a limited capacity
to deal with this damage, possessing a
truncated base excision repair pathway
characterized by only the first enzyme in this
process, -oxoguanine DNA
glycosylase (OGG ), but none of the other downstream elements of
this DNA repair pathway 0 .
As a result, spermatozoa are capable of
responding to oxidative DNA damage by
creating an abasic site at the oxidized base position, but cannot progress the DNA repair any
further (Figure . ).
In contrast, the oocyte possesses very
low levels of OGG but does possess the downstream components of the base excision repair
pathway 0 .
The male and female germ lines therefore
collaborate in resolving any oxidative DNA damage brought into the egg by the fertilizing
spermatozoon. However, if the
fertilizing spermatozoon has particularly
high levels of OHdG, as is
often the case in the subfertile
population 0 , then these residues will remain associated with the chromatin of
the sperm following fertilization and,
because the oocyte is so poorly endowed
with OGG , the residues will persist into the S-phase of the first
mitotic division (Figure . ).
At this point the high mutagenic potential
of OHdG, particularly its
ability to create GC AT transversions, will have a major impact on
the mutagenic load carried by the
embryo 0
. Some measure of protection
against such an eventuality has been
recently indicated in a study 0 reporting an
increase in oocyte OGG activity following fertilization as a consequence of post-translational
modifications to oocyte enzymes
involved in the base excision repair
pathway, causing nuclear localization and
accelerated OHdG excision.
Notwithstanding such changes within the
fertilized egg, the low levels of OGG characteristic of the oocyte emphasize the
vulnerability of embryonic development
to high levels of OHdG carried into the oocyte by the
spermatozoon (Figure . ).
This phenomenon has major implications for
the assisted conception industry, where
potentially damaged spermatozoa are being used to create embryos through the increasingly
popular practice of ICSI. 0
https:/www.cambridge.org/core/terms. https://doi.org/ 0. 0 / .00 Downloaded from
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, subject to the Cambridge Core terms of use, available at Chapter : Physiological and Pathological Aspects of
Sperm Metabolism Damaged base Oxidatively
damaged base Base excision
pathway AP endonuclease OGG glycosylase
APE /XRCC ’ OH ’ dRP
Spermatozoa possess OGG but
none of the other enzymes in the base
excision repair pathway Oocytes possess
very little OGG but do express abundant APE and XRCC
, the downstream contributors to
the base excision repair pathway OGG APE XRCC Abasic site A OGG B
Figure . Vulnerability of embryonic development to
oxidative DNA damage in the sperm nucleus. A, spermatozoa possess only the first enzyme in the base excision repair
pathway, OGG , but none of the other
downstream elements of this repair process. As a result, spermatozoa are capable of responding to
oxidative DNA damage only by the generation of an abasic site. B, oocytes
possess the downstream elements of the
base excision repair pathway, APE and XRCC
, in abundance. Thus under ideal conditions, male and female gametes collaborate to repair oxidative DNA
damage brought into the egg by the fertilizing spermatozoon. However, the lack
of OGG in oocytes
means that any OHdG residues that have
not been dealt with in the spermatozoon will persist into the S-phase of the
first mitotic division, with potential
mutagenic consequences for the embryo. (A black and white version of this
figure will appear in some formats. For the
colour version, please refer to the plate section.) Conclusions
Patterns of sperm metabolism exhibit considerable variation between species, even within the
Eutheria. While glycolysis is clearly
preferred by some species, such as the
human and mouse, as the major means of
generating the ATP needed to support motility,
other species depend heavily upon OXPHOS. While the latter is more efficient from an ATP-generation
perspective, heavy dependence on
mitochondrial function brings with it
risks associated with the attendant generation
of ROS. The latter are particularly pernicious
as far as spermatozoa are concerned, damaging the DNA in the sperm nucleus and mitochondria
while simultaneously triggering lipid
peroxidation cascades that compromise
the fertilizing potential of these cells.
Understanding the origins of the oxidative stress that plagues mammalian spermatozoa and
developing methods of alleviating this
stress are major tasks for future research in this area.
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