Biochem. J. (2011) 436, 687–698 (Printed in Great Britain)
687
doi:10.1042/BJ20110114
Phosphoinositide 3-kinase signalling pathway involvement in a truncated
apoptotic cascade associated with motility loss and oxidative DNA damage
in human spermatozoa
Adam J. KOPPERS1 , Lisa A. MITCHELL1 , Ping WANG, Minjie LIN and R. John AITKEN2
Discipline of Biological Sciences and ARC Centre of Excellence in Biotechnology and Development, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan,
NSW 2308, Australia
Human spermatozoa are characterized by poor functionality
and abundant DNA damage that collude to generate the high
incidences of male infertility and miscarriage seen in our species.
Although apoptosis has been suggested as a possible cause of poor
sperm quality, the ability of these cells to enter an apoptotic state
and the factors that might trigger such an event are unresolved.
In the present study we provide evidence that the commitment
of these cells to apoptosis is negatively regulated by PI3K
(phosphoinositide 3-kinase)/AKT. If PI3K activity is inhibited,
then spermatozoa default to an apoptotic cascade characterized
by rapid motility loss, mitochondrial reactive oxygen species
generation, caspase activation in the cytosol, annexin V binding
to the cell surface, cytoplasmic vacuolization and oxidative
DNA damage. However, the specialized physical architecture
of spermatozoa subsequently prevents endonucleases activated
during this process from penetrating the sperm nucleus and
cleaving the DNA. As a result, DNA fragmentation does not
occur as a direct result of apoptosis in spermatozoa as it does in
somatic cells, even though oxidative DNA adducts can clearly be
detected. We propose that this unusual truncated apoptotic cascade
prepares spermatozoa for silent phagocytosis within the female
tract and prevents DNA-damaged spermatozoa from participating
in fertilization.
INTRODUCTION
in the conventional sense. On the other hand, there have been
numerous sporadic reports of these cells exhibiting some of the
hallmarks of apoptosis, particularly in cases of sub-fertility. For
example, the proportion of spermatozoa exhibiting high levels of
PS (phosphatidylserine) externalization has been correlated with
semen quality, as reflected by the morphology, motility and fertilizing potential of these cells [8–10]. In addition, numerous activated caspases have been reported in human spermatozoa, localized
primarily in the postacrosomal region of these cells (caspases 8, 1
and 3) or, in the case of caspase 9, in the sperm midpiece [11]. Furthermore Kotwicka et al. [12] highlighted a significant correlation
between the presence of activated caspase 3 in human spermatozoa and PS externalization. A third hallmark of apoptosis to have
been detected in human spermatozoa is DNA strand breakage,
frequently in association with signs of oxidative stress [13,14].
Although the appearance of such apoptotic markers in spermatozoa from a variety of species is associated with defective sperm
function and poor pregnancy outcomes, it is not known whether this process is invariably initiated in the testes, or
whether these cells are competent to undergo apoptosis following
ejaculation. The answer to this question has a direct bearing
on the origins of DNA damage seen in mammalian spermatozoa
and the strategies that might be adopted to ameliorate this
damage and reduce the mutational load subsequently carried by
Defective sperm function is the single most common defined
cause of human infertility [1]. Furthermore, the DNA damage
commonly associated with defective human spermatozoa is
known to have a significant impact on fertility, rates of
miscarriage, and the health and wellbeing of the offspring
following assisted conception [2,3]. Given this background
and the high current rates of recourse to assisted reproductive
technologies, insights into the aetiology of DNA damage and
defective functionality in the male germ line are urgently needed.
Apoptosis is a major developmental mechanism that is known to
play a key role in normal spermatogenesis, the testicular response
to toxic injury and the pathogenesis of male infertility [4–7]. It
has also been suggested that the DNA damage that features so
prominently in human spermatozoa is the result of an abortive
apoptotic process that was initiated early in spermatogenesis but
failed to run to completion because the extensive remodelling
of germ cells during spermiogenesis removes the intracellular
machinery needed to effect cell death [6].
Whether mature human spermatozoa can exhibit some or all of
the features of an apoptotic cell is still unresolved. On the one
hand, the fact that these cells are transcriptionally and translationally silent means that they cannot undergo programmed cell death
Key words: apoptosis, DNA damage, mitochondrion, phosphoinositide 3-kinase (PI3K), reactive oxygen species (ROS),
spermatozoon.
Abbreviations used: AIFM, apoptosis-inducing factor, mitochondrion-associated; BAD, Bcl-2-associated death promoter; BWW, Biggers–Whitten–
Whittingham; CAD, caspase-activated DNase; CASA, computer-assisted sperm analysis; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DIABLO,
direct IAP (inhibitor of apoptosis)-binding protein with low pI; DTT, dithiothreitol; ENDOG, endonuclease G; FLICA, fluorescently labelled inhibitor of caspase
assay; 8OHdG, 8-hydroxy-2 -deoxyguanosine; PI, propidium iodide; PI3K, phosphoinositide 3-kinase; PIP3 , phosphatidylinositol (3,4,5)-trisphosphate; PS,
phosphatidylserine; PTEN, phosphatase and tensin homologue deleted on chromosome 10; ROS, reactive oxygen species; SMAC, second mitochondrialderived activator of caspase; TBS, Tris-buffered saline; TBST, TBS containing 0.01 % Tween 20; TdT, deoxynucleotidyltransferase; TUNEL, terminal TdT
(deoxynucleotidyltransferase)-mediated dUTP nick-end labelling.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
688
A. J. Koppers and others
the embryo [15]. In the present paper we report, for the first
time, that these highly specialized cells, lacking both cell-cycle
checkpoints and any form of transcriptional activity, can default to
a truncated intrinsic apoptotic cascade, characterized by motility
loss and oxidative DNA damage, following dephosphorylation
of PI3K (phosphoinositide 3-kinase) and AKT. Furthermore this
process may be clearly distinguished from apoptosis in somatic
cells because the physical architecture of these cells prevents the
physical translocation of nucleases from the mitochondria and
cytoplasm to the sperm nucleus. These results explain why the
promotion of PI3K activity is so critical to sperm survival and
why DNA damage in spermatozoa is largely oxidative [14,16].
MATERIALS AND METHODS
Chemicals and culture medium
Unless stated otherwise, all chemicals were purchased from
Sigma–Aldrich, whereas all fluorescent probes were purchased
from Molecular Probes. Spermatozoa were cultured in BWW
(Biggers–Whitten–Whittingham) medium supplemented with
0.1 % polyvinyl alcohol, 5 units/ml penicillin and 5 mg/ml
streptomycin [17]. The antibodies used in the present study were
all from Abcam, with the exception of the anti-phospho-AKT
(Thr308 ) antibody, which was purchased from Genesearch, and
the anti-PTEN (phosphatase and tensin homologue deleted on
chromosome 10) antibodies (anti-PTEN and anti-phospho-PTEN,
pSer380 ), which were purchased from Calbiochem. The final
concentrations at which the various antibodies were used were as
follows: anti-phospho-PI3K targeting Tyr467 and Tyr199 (1:500),
anti-PI3K (1:100), anti-phospho-AKT antibodies targeting
pThr308 (1:20), pThr34 (1:20), pSer124 (1:20) and pSer473 (1:20)
respectively, anti-pan-AKT1 and anti-pan-AKT2 (1:50), antiAIFM (apoptosis-inducing factor, mitochondrion-associated)
(1:500) anti-cytochrome c (1:50), anti-DIABLO [direct IAP
(inhibitor of apoptosis)-binding protein with low pI] homologue
(Drosophila) {DIABLO [SMAC (second mitochondrial-derived
activator of caspase)]} (1:50), anti-ENDOG (endonuclease G)
(1:50), anti-phospho-BAD (Bcl-2-associated death promoter)
targeting Ser99 (1:50), anti-PTEN (1:50), monoclonal antiphospho-PTEN targeting pSer380 (1:50) and anti-CAD (caspaseactivated DNase) (1:50). Although we did not independently
validate these commercially available antibodies, transcripts for
all of these proteins are known to be present in the male germ
line at the late spermatid stage of development when mRNA
is still relatively abundant (http://mrg.genetics.washington.edu).
In addition, previous studies have confirmed the presence of
CAD, cytochrome c, DIABLO(SMAC) and AIFM in male germ
cells, including spermatozoa [18–22]. For all antibodies, negative
controls were run comprising the secondary antibody alone.
Preparation of human spermatozoa
Institutional and State Government ethical approval was secured
for the use of donated human semen samples for the purposes
of this research. Samples from unselected normozoospermic
donors of unknown fertility were collected into sterile containers
prior to immediate transportation to the laboratory. Following
initial inspection for liquefaction, consistency, debris and volume,
assessments of cell count and motility were conducted and cell
viability was measured using the eosin exclusion test [23]. After
allowing at least 30 min for liquefaction to occur, the spermatozoa
were fractionated on a discontinuous two-step Percoll gradient
(90 %/45 %), as described previously [24]. The spermatozoa used
in these experiments were recovered from the base of the high
c The Authors Journal compilation c 2011 Biochemical Society
density portion of the gradient. These cells were then washed with
5 ml of BWW, centrifuged at 600 g for 15 min and finally resuspended in BWW. Contaminating leucocytes were removed from
all samples using magnetic Dynabeads coated with a monoclonal
antibody directed against the common leucocyte antigen CD45
(Dynal) and confirmed using a zymosan provocation assay [24].
SDS/PAGE and Western blot analysis
Following treatment, spermatozoa were centrifuged (600 g for
5 min) and protein was extracted by solubilizing the cells
in an SDS extraction buffer [2 % SDS and 10 % sucrose in
0.1875 M Tris/HCl (pH 6.8)] containing a protease inhibitor
cocktail (Roche) for 5 min at 100 ◦ C. The spermatozoa were
then centrifuged at 10 000 g for 15 min in order to remove
insoluble components. Quantification of the isolated protein
supernatant was achieved using a BCA (bicinchoninic acid)
protein assay kit (Pierce) according to the manufacturer’s
instructions. Equal amounts of total protein (5 μg, equivalent to
approximately 750 000 cells) were boiled in SDS/PAGE sample
buffer (SDS extraction buffer as above supplemented with 2 % 2mercaptoethanol and Bromophenol Blue) for 5 min and resolved
on 10 % polyacrylamide gels. The resolved proteins were then
transferred on to nitrocellulose membranes under a constant
current of 300 mA for 1 h.
Nitrocellulose membranes were blocked overnight at 4 ◦ C with
3 % BSA (Research Organics) in TBS (Tris-buffered saline:
100 mM Tris/HCl, pH 7.6, and 150 mM NaCl) (pH 7.4)
supplemented with 0.1 % Tween 20. Membranes were rinsed
in TBS and then probed with the appropriate concentration of
primary antibodies in 1 % BSA and 0.1 % Tween 20 in TBS for 2 h
at room temperature (22 ◦ C). Following incubation, membranes
were washed three times in TBST (TBS containing 0.01 % Tween
20) for 10 min. Membranes were then further probed for 1 h with
a 1:1000 dilution of HRP (horseradish peroxidase)-conjugated
secondary antibody at room temperature. Following a further
three washes in TBST, cross-reactive proteins were visualized
using an ECL (enhanced chemiluminescence) kit (GE Healthcare)
according to the manufacturer’s instructions.
Immunolocalization of proteins on fixed spermatozoa
Following treatment, spermatozoa were fixed in 4 % paraformaldehyde, washed three times with PBS, plated on to poly-L-lysinecoated glass slides and placed in a humid chamber at 37 ◦ C. The
cells were permeabilized with 0.2 % Triton X-100 for 15 min,
rinsed with PBS and blocked with 10 % serum/3 % BSA for
1 h. Slides were then washed three times with PBS for 5 min
and incubated in a 1:50 dilution of primary antibody overnight
at 4 ◦ C, before being subjected to three 5 min washes with PBS
and incubation in a 1:100 dilution of FITC-conjugated secondary
antibody for 2 h at 37 ◦ C. Slides were again washed three times for
5 min and mounted in 10 % Mowiol 4-88 (Calbiochem) with 30 %
glycerol in 0.2 M Tris/HCl (pH 8.5) with 2.5 % DABCO [1,4diazobicyclo-(2.2.2)-octane]. Spermatozoa were then imaged on
a Zeiss LSM510 confocal microscope (Carl Zeiss) using argon
laser excitation (488 nm) with emission collection at 500–530 nm
(green).
MitoSOX Red assay
Intracellular generation of mitochondrial superoxide anion was
determined using MitoSOX Red (Molecular Probes). For this
assay, MitoSOX Red (5 mM in DMSO) and the cell viability stain
SYTOX Green (0.125 mM in DMSO) stock solutions were diluted
in BWW and added to spermatozoa at 1 × 107 cells/ml to give
Truncated apoptosis in human spermatozoa
final concentrations of 2 mM and 0.05 mM respectively. After an
incubation period of 15 min at 37 ◦ C, samples were centrifuged
for 5 min at 600 g; the pellets were then resuspended in 1 ml
of BWW and transferred to a 5 ml FACS tube. The MitoSOX
Red (red) and SYTOX Green (green) fluorescence was finally
measured on a FACSCalibur flow cytometer (Becton Dickinson).
Argon laser excitation at 488 nm was coupled with emission
measurements using 530/30 band pass (green) and 585/42 band
pass (red) filters. Non-sperm-specific events were gated out and
10 000 cells were examined. The results were expressed as the percentage of cells that were alive and stained positively with the
probe. Non-viable cells were excluded from the analysis because
commercial preparations of MitoSOX Red contain trace amounts
of ethidium bromide that can generate spurious signals if the
integrity of the plasma membrane is compromised in any way.
JC-1 assay
JC-1 (Molecular Probes) is a fluorescent dye that can report on the
state of the mitochondrial membrane potential (). JC-1 stock
solution (7.5 mM in DMSO) was diluted in BWW and added to
spermatozoa at 1 × 107 cells/ml to give a final concentration of
2 mM. For a positive control, cells were incubated for 15 min
with 10 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone)
prior to JC-1 staining. After incubation with JC-1 for 15 min, cells
were centrifuged (600 g) and the pellet was resuspended in 1 ml
of BWW. This suspension was then transferred to a 5 ml FACS
tube, where PI (propidium iodide) (10 μg/ml) was added just prior
to analysis. The JC-1 and PI fluorescence was then measured on
a FACSCalibur flow cytometer (Becton Dickinson). Argon laser
excitation at 488 nm was coupled with emission measurements
using >670 long-pass filter (far red; PI) to exclude dead cells
from analysis, followed by 530/30 band pass (green; low )
and 585/42 band pass (red; high ) filters. Non-sperm-specific
events were gated out and 10 000 cells were examined.
CASA (computer-assisted sperm analysis)
Sperm motility parameters were analysed by CASA using
a Hamilton Thorne Version 12 IVOS (Hamilton Thorne
Biosciences). For each sample measurement, a 2.5 μl aliquot
of spermatozoa was loaded on to a standard four-chamber slide
(Leja). A minimum of 200 spermatozoa were examined for each
sample using standard settings (30 frames acquired at a frame rate
of 60 Hz and a temperature of 37 ◦ C in 20 μm-deep chambers).
Samples were analysed for their percentage standard motility,
as well as progressive motility (average path velocity>25 μm/s;
straightness>75 %). Motility data were expressed as a percentage
of an untreated control sample such that the 0 μM wortmannin
category represented the impact of the vehicle alone.
Measurement of activated caspase
FLICA (fluorescently labelled inhibitor of caspase assay;
Immunochemistry Technologies) was used to measure active
pan-caspases in whole cells. The FLICA dye was stored as a
150× stock solution in DMSO, according to the manufacturer’s
instructions. This stock solution was diluted in BWW and added
to spermatozoa to give a final concentration of 1×FLICA dye.
Spermatozoa were incubated for 1 h with the dye, and in the
last 15 min SYTOX Green was added at a final concentration of
0.05 mM, in order to monitor cell viability. This was followed
by centrifugation for 5 min at 600 g and re-suspension in 1 ml of
BWW. Flow cytometric analysis was performed as described for
the MitoSOX Red Assay.
689
Annexin V externalization
The externalization of PS from the inner-to-outer leaflet of the
plasma membrane is a feature of apoptosis. The annexin V–FITC
apoptosis detection kit (Sigma–Aldrich) allows the detection of
PS on the cell surface through FITC-conjugated annexin V. The
addition of a cell-viability indicator, PI, allows the differentiation
between apoptotic cells (annexin V positive, PI negative), necrotic
cells (annexin V positive, PI positive) and viable cells (annexin
V negative, PI negative). For this analysis, spermatozoa were
centrifuged (600 g for 5 min), and the pellet was resuspended
in buffer containing FITC-conjugated annexin V according the
manufacturer’s instructions. Spermatozoa were incubated with
the antibody for 1 h prior to centrifugation at 600 g for 5 min
and re-suspension in 1 ml of BWW. Just prior to flow cytometric
analysis, PI (stored in PBS) was added the samples to give a final
concentration of 0.01 mg/ml. The annexin V and PI fluorescence
was then measured on a FACSCalibur flow cytometer (Becton
Dickinson). Argon laser excitation at 488 nm was coupled with
emission measurements using a >670 nm long-pass filter (far red;
PI) to exclude dead cells from the analysis, followed by 530/30 nm
band pass (green) filters. Non-sperm-specific events were gated
out and 10 000 cells were examined.
Immunofluorescence assay for 8OHdG
(8-hydroxy-2 -deoxyguanosine)
The formation of 8OHdG was measured using a specific antibody
(Biotrin OxyDNA Test Kit, Biotrin International) conjugated to
FITC. For the positive control, spermatozoa were incubated for
1 h with H2 O2 (2 mM) and FeCl2 ·4H2 O (1 mM) in a final volume
of 200 μl of BWW. The H2 O2 concentration was determined
by measuring the absorbance at 240 nm. The cells were then
washed twice in BWW, resuspended in 100 μl of 2 mM DTT
(dithiothreitol) in BWW and incubated for 45 min at 37 ◦ C to
relax the chromatin. After centrifugation at 600 g for 5 min, the
cells were fixed by resuspending the pellet in 200 μl of 2 %
paraformaldehyde and incubating at 4 ◦ C for 15 min. The cells
were subsequently washed in PBS and stored in 200 μl of 0.1 M
glycine at 4 ◦ C for a maximum of 1 week. Fixed cells were
ultimately washed and resuspended in 100 μl of 0.2 % Triton X100. Cells were then incubated at room temperature for 15 min,
centrifuged (600 g for 5 min) and washed in wash solution (from
the Biotrin OxyDNA Test Kit); 50 μl of blocking solution (3 %
BSA) was then added before incubation at 37 ◦ C for 1 h. The anti8OHdG antibody was further purified by adding approximately
1 mg of activated charcoal powder, incubated at room temperature
for 1 h and centrifuged twice at 600 g for 5 min. The supernatant
containing the purified antibody was then added at a 1:50 dilution
to the fixed cells in enough wash solution to achieve a final volume
of 100 μl. This was incubated for 1 h and, finally, the cells were
washed twice, resuspended in 1 ml of PBS and transferred to 5 ml
FACS tubes for flow cytometry analysis. Fluorescence was then
measured on a FACSCalibur flow cytometer (Becton Dickinson);
argon laser excitation at 488 nm was coupled with emission
measurements using 530/30 nm band pass (green). Non-spermspecific events were gated out and 10 000 cells were examined.
TUNEL [terminal TdT (deoxynucleotidyltransferase)-mediated
dUTP nick-end labelling] assay
DNA cleavage may yield double-stranded and singlestranded DNA breaks. Both types of break can be detected by
labelling the free 3 -OH terminal with fluorescent nucleotides
(fluorescein-labelled dUTPs) in an enzymatic reaction catalysed
by TdT in the TUNEL assay. Following treatment, spermatozoa
c The Authors Journal compilation c 2011 Biochemical Society
690
A. J. Koppers and others
were centrifuged (600 g for 5 min) and resuspended in 2 mM
DTT in BWW for 45 min to relax the chromatin [25]. After
centrifugation at 600 g for 5 min, the cells were fixed by
resuspending the pellet in 2 % paraformaldehyde and then
incubating at 4 ◦ C for 15 min. The cells were subsequently washed
in PBS and stored in 200 μl of 0.1 M glycine at 4 ◦ C. For
analysis, spermatozoa were centrifuged (600 g for 5 min) before
resuspending the pellet in 100 μl of permeabilization solution
(0.02% Triton X-100 in PBS) and incubating for 2 min at 4 ◦ C.
The cells were then centrifuged (600 g for 5 min) and the pellets
resuspended in PBS. The positive control was treated with 100 μl
of DNase I (1 mg/ml) and 10 μl of MgSO4 (10 mM) for 30 min
at 37 ◦ C. TUNEL labelling was achieved with the In Situ Cell
Death Detection Kit (Roche Applied Science) according to the
manufacturer’s instructions. Cells were then washed twice in PBS,
diluted to a final volume of 500 μl in PBS and kept in the dark
for analysis using flow cytometry. Flow cytometric analysis was
performed as described for the 8OHdG assay.
Ultrastructure
Spermatozoa were centrifuged at 1000 g for 10 min, and then
resuspended and fixed in 500 μl of 2 % formaldehyde/2.5 %
glutaraldehyde in 0.1 M PBS (pH 7.3) for 2 h at 4 ◦ C. To
remove the fixative, spermatozoa were washed three times by
centrifugation at 1000 g for 5 min and resuspended in 0.1 M
PBS. After centrifugation, the spermatozoa were submitted to
successive dehydration steps. Cells were immersed progressively
in increasingly concentrated ethanol solutions (v/v) at 50 %,
75 % and 95 % for 5 min each, then in three changes of 100 %
ethanol for 5 min each, and in two changes of 100 % acetone
for 5 min. Then, successive Spurr’s resin filtration steps were
carried out by immersing the dehydrated samples in progressively
more concentrated resin solution in acetone (v/v), beginning with
33 % and 50 % resin for 1 h each at room temperature, then
66 % and 100 %, each overnight at room temperature, and finally
transferring to fresh 100 % resin and polymerizing for 24 h at
60 ◦ C in a laboratory oven.
Sections (70 nm) of the embedded tissue were cut on an Ultracut
E ultramicrotome (Reichert-Jung), floated out on Milli-Q water
and transferred on to 100-mesh copper grids. Grids were then
stained with 0.5 % uranyl acetate in 30 % ethanol for 10 min
and rinsed with distilled H2 O; further staining was achieved with
lead citrate for 10 min. Lead precipitates on grid sections were
removed by rinsing in 0.05 M NaOH before further rinsing in
distilled H2 O. Grids were left at room temperature to dry and the
stained sections were examined using a JEOL-1200EX electron
microscope operating at 80 kV.
Statistical analysis
All experiments were repeated at least three times on independent
samples and the results were analysed by ANOVA or a paired
Student’s t test using the SuperANOVA and Statview programs
respectively (Abacus Concepts); post hoc comparison of group
means was by Fisher’s protected least significant difference test.
Differences with a P value of <0.05 were regarded as significant.
RESULTS
Elements of the PI3K signalling complex are localized to specific
domains in human spermatozoa
Western blot analyses using anti-AKT and anti-PI3K (p85
subunit) polyclonal antibodies clearly demonstrated the presence
of these key enzymes in human spermatozoa (Figure 1A).
c The Authors Journal compilation c 2011 Biochemical Society
Immunocytochemical studies not only confirmed their presence
in human spermatozoa, but also revealed highly specific patterns
of subcellular localization. Thus an antibody against the p100 kDa
catalytic subunit of PI3K revealed this enzyme to be located in the
principal piece of the sperm tail, the neck and the acrosome, but to
be excluded from the midpiece of the cell where the mitochondria
and residual cytoplasm are located (Figure 1B, indicated with
arrows). An antibody against the phosphorylated form of the
p85 regulatory subunit of PI3K, targeting phospho-Tyr467 and
phospho-Tyr199 , also revealed the activated form of this component
of the heterodimer to be largely located in the principal piece of
the sperm tail and absent from the midpiece (Figure 1C).
In most cells the ability of PI3K to generate a powerful
signalling molecule, PIP3 [phosphatidylinositol (3,4,5)-trisphosphate], is in dynamic equilibrium with a phosphatase that
negatively regulates the bioavailability of PIP3 by catalysing
its dephosphorylation to the corresponding bisphosphate, PIP2
[phosphatidylinositol (4,5)-bisphosphate]. This phosphatase is
known as PTEN. Immunocytochemical localization of PTEN
generated a very weak signal, predominantly in the midpiece
of the cell (Figure 1D). By contrast, an antibody against
the phosphorylated, stabilized, form of this enzyme (pSer380 ),
indicated that a majority of the PTEN in spermatozoa exists in
a phosphorylated state and is localized in the equatorial segment
of the sperm head, distant from the PI3K in the principal piece of
the tail (Figure 1E). This physical separation of PI3K and phosphorylated PTEN is absolutely unique to spermatozoa and would
ensure that the former is free to generate PIP3 without any
interference from PTEN phosphatase activity. In keeping with
this conclusion we found that addition of the inhibitor bpV
(a general vanadate-based kinase inhibitor with demonstrable
activity against PTEN) [26] did not promote the pro-survival
effects of prolactin or insulin on spermatozoa (results not shown),
and actually induced a dose-dependent suppression of motility and
progressive motility (Figure 1F), rather than the enhancement of
motility that might have been anticipated if PTEN suppression
had led to a corresponding increase in PI3K activity.
A major target for PIP3 is the serine/threonine kinase AKT.
Two of the three major isoforms of AKT expressed in the testes
are AKT1 (RAC-α serine/threonine-protein kinase), a recognized
inhibitor of apoptosis and AKT2 (RAC-β serine/threonine-protein
kinase), an important constituent of the insulin signalling pathway.
Immunocytochemistry revealed that AKT1 was strongly present
throughout the sperm tail, including the principal piece and the
midpiece where the machinery for apoptosis is located, in the form
of mitochondria and the only significant quantity of cytoplasm
present in this cell type (Figures 2A and 2B). AKT2 was also
strongly present in the midpiece of the cell and, to a lesser
extent, the proximal part of the flagellar principal piece; however,
in contrast with AKT1, AKT2 also gave a very strong signal in
the sperm head, particularly in the vicinity of the acrosome
(Figure 2C). The activated phosphorylated forms of this kinase
including pThr34 -AKT (present in AKT1 but not AKT2) as well as
pThr308 -, pSer473 - and pSer124 -AKT (represented in both AKT1 and
AKT2) were also detectable in human spermatozoa. Significantly,
all antibodies exhibited strong staining in the sperm midpiece, in
close proximity to the sperm mitochondria (particularly pThr34
and pThr308 ; Figures 2D and 2E). In addition, pThr308 gave a
particularly strong signal in the acrosome (Figure 2E), whereas
anti-pThr473 also gave a signal along the principal piece of the tail
(results not shown).
In order to determine whether PIP3 generated by PI3K in the
principal piece of the sperm tail could be responsible for activating
AKT in this and other regions of the cell, the PI3K inhibitor
wortmannin was used. Addition of wortmannin led to the highly
Truncated apoptosis in human spermatozoa
Figure 1
691
Presence of the PI3K, AKT and PTEN in human spermatozoa
(A) Demonstration that both PI3K (regulatory subunit) and AKT are present in human spermatozoa by Western blot analysis. The molecular mass in kDa is indicated on the right-hand side.
(B) Immunocytochemical localization of PI3K (p110 catalytic subunit) to the principal piece, the neck and the acrosomal region of human spermatozoa. Note the absence of staining in the midpiece
where the mitochondria are located (indicated by an arrow). (C) Immunocytochemical localization of the phosphorylated activated form of PI3K (p85 regulatory subunit) to the principal piece;
again note the lack of staining in the midpiece (indicated by an arrow). (D) Weak immunocytochemical signal generated by PTEN in sperm midpiece. (E) A majority of the PTEN is stabilized by
phosphorylation and located in the equatorial segment of the sperm head. This physical separation of PI3K and PTEN is unique to spermatozoa and ensures that the former is maximally active. Scale
bar in (B)–(E) = 5 μm. (F) The physical separation of PI3K and PTEN also explains why an inhibitor of PTEN, bpV, which should support sperm function by enhancing PI3K activity, is either inactive
or, at high concentrations, inhibitory. Results are means +
− S.E.M.
effective suppression of phospho-AKT expression (Figure 2A). A
major downstream target of AKT kinase activity, BAD, is a wellcharacterized regulator of apoptosis, so the status of this protein
was also examined before and after exposure to wortmannin.
Using an antibody that could recognize the phosphorylated form
of BAD (pSer99 ) by immunocytochemistry (but not Western blot
analysis), we demonstrated that although phospho-BAD could
clearly be detected in the midpiece and tail of control spermatozoa
(Figure 2F), treatment with wortmannin led to complete loss of
the pSer99 epitope (Figure 2F, bottom panels), indicating a clear
change in the phosphorylation status of this apoptosis mediator.
Since such a change in BAD phosphorylation would be
expected to cause this protein to adopt a pro-apoptotic role,
we next looked for other signs of an intrinsic apoptotic
cascade in wortmannin-treated spermatozoa, including changes
in sperm motility, vitality, mitochondrial ROS (reactive oxygen
species) generation, mitochondrial membrane potential (), PS
exposure, caspase activation and DNA damage.
Suppression of PI3K leads to a loss of motility
One of the most obvious and rapid changes to occur following
the induction of apoptosis with wortmannin was a highly
significant dose- (P < 0.001) and time- (P < 0.001) dependent
loss of the percentage of sperm motility and progressive motility
(Figures 3A–3D). Thus a combination of dose–response analyses,
initially covering a broad array of concentrations (Figures 3A
and 3C), followed by a more focused and limited range,
demonstrated that within 4 h both motility and progressive
motility were significantly suppressed by exposure to >5 μM
wortmannin (Figures 3B and 3D) and, at doses above 20 μM,
motility was effectively lost and cell vitality exhibited a dosedependent decline (P < 0.001; Figure 4A). In addition, parallel
studies with another PI3K inhibitor, LY294002, revealed the
same highly significant dose- and time-dependent inhibition of
sperm movement (P < 0.001) and vitality (P < 0.01), reinforcing
the notion that suppression of this kinase causes spermatozoa to
default to a pathway leading to cell death.
Suppression of PI3K leads to mitochondrial ROS generation
In order to determine whether the loss of motility and vitality
observed after disruption of the PI3K/AKT/BAD axis with
wortmannin involved the activation of apoptosis in mature
human spermatozoa, these cells were examined for activities
characteristic of cell entry into an intrinsic apoptotic pathway. As
c The Authors Journal compilation c 2011 Biochemical Society
692
Figure 2
A. J. Koppers and others
Impact of wortmannin on AKT and BAD expression
(A) Western blot analysis indicating the presence of phospho-AKT (top panel) in human spermatozoa and demonstrating the dephosphorylation of this kinase in the presence of the PI3K inhibitor
wortmannin; the loading control shown in the bottom panel was an anti-pan-AKT antibody. The molecular mass in kDa is indicated on the right-hand side. (B) Immunocytochemical analyses revealed
the presence of AKT1 along the entire length of the sperm tail including the neck, midpiece and principal piece, whereas (C) AKT2 was principally located in the sperm acrosome and the midpiece,
where the mitochondria are located. (D) The phosphorylated form of AKT1 (pThr34 ) generated a strong signal in the sperm tail with clear emphasis on the midpiece, whereas (E) pThr308 , (present
in both AKT1 and AKT2) was found in the midpiece and in the acrosomal region. (F) The anti-apoptotic factor phospho-BAD was predominantly located in the midpiece of human spermatozoa
(indicated by an arrow); however, all cross-reactivity was lost in the presence of 20 μM wortmannin, suggesting the dephosphorylation of this apoptosis regulator. Scale bars = 5 μm.
illustrated in Figure 4(B), the addition of wortmannin to human
spermatozoa in vitro resulted in significant dose- (P < 0.001) and
time- (P < 0.05) dependent increases in mitochondrial ROS generation, as measured by MitoSOX Red. Mitochondrial ROS
generation in live cells increased with wortmannin doses up to
20 μM, but collapsed at higher concentrations as the spermatozoa
lost their vitality. Since a loss of is also a common
signature of apoptotic cells that has been observed to accompany
mitochondrial ROS generation [27], we also examined the effect
of wortmannin on this aspect of mitochondrial activity. Use of
the fluorescent marker JC-1 revealed that, at wortmannin doses
where mitochondrial ROS generation was active and cell vitality
was not compromised (20 μM), wortmannin had no effect on
, even after 24 h exposure (Figure 4C). Parallel studies with
LY294002 confirmed that the inhibition of PI3K activity resulted
in dose- (P < 0.001) and time- (P < 0.001) dependent increases
in mitochondrial ROS generation, in the absence of a significant
collapse of mitochondrial membrane potential (results not shown).
These results suggested that the rapid stimulation of mitochondrial
ROS was not due to an uncoupling mechanism, nor was the
latter an early component of this apoptotic pathway in human
spermatozoa.
c The Authors Journal compilation c 2011 Biochemical Society
Suppression of PI3K activates the intrinsic apoptotic pathway
in human spermatozoa
In order to determine whether the suppression of PI3K/AKT
resulted in the expression of additional hallmarks of apoptosis
other than mitochondrial ROS generation, these cells were
also examined for PS externalization and caspase activation.
Wortmannin was found to result in highly significant time(P < 0001) and dose- (P < 0001) dependent increases in PS
externalization on the surface of viable human spermatozoa
(Figure 4D). PS externalization appeared to be a relatively late
event in sperm apoptosis because significant changes were only
observed after 24 h, at which point the response was found
to increase with wortmannin doses up to 20 μM and then
declined in concert with the progressive loss of cell viability
(Figure 4A). Using FLICA, wortmannin was also shown to
induce a highly significant (P < 0.001) dose-dependent increase
in caspase activation in human spermatozoa (Figure 5A). In order
to determine the temporal relationship between PS externalization
and caspase activation, a time-course analysis was conducted
following the activation of apoptosis with wortmannin. The
results of this study again emphasized that PS externalization
Truncated apoptosis in human spermatozoa
Figure 3
Inhibition of PI3K leads to the effective disruption of sperm motility
(A) Dose- and time-dependent inhibition of sperm motility with wortmannin over a wide dose
range. (B) Using a more refined range of concentrations, significant effects of wortmannin on
sperm motility were evident at 4 h with 5 μM of this inhibitor. (C) CASA analysis of progressive
motility (average path velocity > 25 μm/s; straightness > 75 %) revealed a similar dose- and
time-dependent inhibition of sperm movement. (D) As with overall motility, a dose–response
analysis utilizing a more limited range of concentrations revealed that progressive motility was
significantly suppressed with >5 μM wortmannin over a 4 h time scale. Results are expressed
as a percentage of an untreated control sample, such that a dose of 0 μM equates with the
vehicle-only control. Results are means +
− S.E.M. (***P < 0.001 and **P < 0.01 compared
with the vehicle-only control).
is a relatively late event in the apoptotic cascade as expressed by
spermatozoa. Thus, although significant caspase activation was
evident 4 h after the initiation of apoptosis (Figure 5B; P < 0.001),
PS externalization was not manifest until 8 h (Figure 5B;
P < 0.001). The involvement of PI3K in the activation of this
apoptotic cascade was also supported by the fact that highly
significant (P < 0.001) changes in caspase activation and PS
externalization (P < 0.001) were also observed when PI3K
activity was suppressed by LY294002 (results not shown).
Interestingly, the addition of other activators of apoptosis in
somatic cells, including staurosporine, lipopolysaccharide, Kdo
and genistein, did not elicit the activation of caspases (Figure 5C),
or induce any significant changes in motility, vitality or 8OHdG
(results not shown) in human spermatozoa over the same time
period, emphasizing the dramatic differences that exist between
the apoptotic pathway(s) expressed in these cells relative to other
cell types.
The appearance of DNA strand breaks is another major
hallmark of an apoptotic cell. Such apoptosis-associated DNA
fragmentation is induced by endonucleases which are either
released from the mitochondrial inter-membranous space, (AIFM
or ENDOG), or activated in the cytosol (CAD), prior to their
693
translocation to the nucleus. The presence of DNA strand breaks
in human spermatozoa has been widely observed in defective
sperm populations and is known to be induced by a wide variety
of stimuli, including various forms of electromagnetic radiation
and xenobiotics [15,28,29]. However, within the time frame of
these studies, the addition of wortmannin was unable to induce
DNA strand breaks as measured by the TUNEL assay, even 24 h
after exposure (Figure 5D).
The reason for this lack of DNA fragmentation became evident
following immunocytochemical localization of three major nucleases in human spermatozoa, CAD (Figures 6A and 6B), ENDOG
(Figures 6C and 6D) and AIFM (Figures 6E and 6F). This analysis
revealed that these effectors of apoptosis were predominantly
located in the sperm midpiece, where the mitochondria and a
majority of the sperm cytoplasm are located. Stimulation of
apoptosis with wortmannin did not induce the translocation of
these nucleases from the sperm midpiece to the nucleus because
the physical architecture of the cell, featuring mitochondria and
residual cytoplasm in one subcellular compartment (midpiece)
and nuclear DNA in another (sperm head), does not permit it.
Other effectors of the apoptotic cascade, such as cytochrome
c (Figures 6G and 6H) or DIABLO(SMAC) (Figures 6I and
6J) were also confined to the midpiece of the cell, before and
after activation of the intrinsic apoptotic cascade with wortmannin.
Recent studies have provided evidence highlighting the clinical
importance of oxidative stress in the aetiology of DNA damage in
human spermatozoa [14]. So, even though the impeded migration
of nucleases from the sperm midpiece to the nucleus precluded
endonuclease-mediated DNA cleavage during apoptosis, the
activation of mitochondrial ROS could have induced oxidative
DNA damage that might, in time, have generated the expected
strand breaks. To examine this possibility the formation of
8OHdG, a marker of oxidative DNA damage, was analysed
following wortmannin exposure. In this analysis, wortmannin was
shown to induce a highly significant (P < 0.001) dose-dependent
increase in the prevalence of oxidative DNA damage in human
spermatozoa after 24 h incubation (Figure 6K).
Finally, we examined whether the induction of this apoptotic
cascade was associated with ultrastructural changes similar to
those seen in somatic cells following the initiation of this process.
Ultrastructural analysis of human spermatozoa, under conditions
that resulted in both caspase activation and PS externalization,
revealed several morphological changes compared with control
cells (Figure 7). Specifically, activation of the intrinsic apoptotic
cascade with wortmannin resulted in the appearance of large
vacuoles and membrane protrusions in the sperm midpiece, where
the machinery for expressing this process (mitochondria and a
significant cytoplasmic space) is located.
DISCUSSION
Several authors have identified PI3K in the spermatozoa of a variety of mammalian and avian species and assigned to this molecule
roles in such cellular processes as the acrosome reaction, capacitation and motility regulation [30–35]. Yet others have identified
AKT and PTEN in this cell type [36–38], supporting the notion
that biochemical pathways involving PI3K, AKT and PTEN are
centrally involved in sperm cell biology. However, the present
study is the first to fully investigate the role of this pathway in the
modulation of apoptosis in mature human spermatozoa. The results of this analysis revealed that wortmannin could successfully
inhibit PI3K phosphorylation and thereby disrupt the ability of
this kinase to phosphorylate its downstream target, AKT, on Thr308
c The Authors Journal compilation c 2011 Biochemical Society
694
Figure 4
A. J. Koppers and others
Changes in vitality, mitochondrial activity and PS externalization in response to PI3K inhibition
Exposure to wortmannin did not have an effect on sperm vitality until doses in excess of 100 μM were reached (*P < 0.05 and ***P < 0.001 compared with the vehicle-only control). (B) At doses
of inhibitor that did not compromise cell vitality (20 μM), a highly significant impact on mitochondrial ROS generation was observed in live cells (SYTOX green negative) with MitoSOX Red in
a dose- and time-dependent manner (*P < 0.05 and **P < 0.01 compared with the vehicle-only control). (C) The induction of mitochondrial ROS production was achieved in the absence of any
significant change in mitochondrial membrane potential as measured with JC-1; as a positive control, incubation with the proton ionophore CCCP was shown to completely silence the JC-1 signal.
(D) Analysis of PS externalization using annexin V in conjunction with PI demonstrated minimal change within 4 h, but a clear dose-dependent response was observed at 24 h, peaking at 20 μM
(*P < 0.05 and ***P < 0.001 compared with the vehicle-only control).
(Figure 2A). The resultant change in AKT kinase activity resulted
in BAD altering from a phosphorylated (anti-apoptotic) to a nonphosphorylated (pro-apoptotic) state (Figure 2F). This change in
the phosphorylation status of BAD is known to allow Bak/Bax
to form pro-apoptotic pores in the outer mitochondrial membrane
and to promote the ability of this molecule to form mitochondrial
pores in its own right [39,40]. As a result, BAD dephosphorylation
was associated with the appearance of several changes that were
consistent with the intrinsic apoptotic cascade observed in somatic
cells, including mitochondrial ROS formation, caspase activation,
PS externalization and cytoplasmic vacuolization [41–43].
Thus the present study demonstrates, for the first time,
that mature functional human spermatozoa can undergo an
intrinsic apoptotic cascade. Indeed the latter appears to be
a default pathway for these cells. As soon as conditions
arise that compromise the phosphorylation status of the
PI3K/AKT complex, then these cells enter an intrinsic apoptotic
pathway associated with the dephosphorylation of BAD and the
induction of mitochondrial permeability. Interestingly, most of
the stimuli that induce apoptosis in somatic cells are completely
without effect in human spermatozoa, including staurosporine,
lipopolysaccharide, Kdo and genistein (Figure 5B). However
some features of apoptosis can apparently be induced in these cells
by exposure to a variety of non-receptor-mediated stimuli that
ultimately create a state of oxidative stress, including exposure to
c The Authors Journal compilation c 2011 Biochemical Society
radiofrequency electromagnetic radiation [44], cryostorage [45],
heat [44] or the direct addition of H2 O2 [46]. As far as we
are aware, there are no reports of receptor-activated apoptosis
in human spermatozoa. The only possible exception could be
the apoptosis-inducing properties of progesterone [46]; however,
this steroid probably exerts its pro-apoptotic effect indirectly
by mobilizing calcium, which then secondarily induces ROS
formation and oxidative stress [47].
Since we can find no evidence to indicate that apoptosis is
actively induced via a receptor-mediated mechanism in mature
human spermatozoa, we propose that these cells revert to
this pathway in response to stress, as a form of programmed
senescence. Although we fully acknowledge that the inhibitor we
have used extensively in the present study, wortmannin, lacks
specificity, it has proven to be a particularly effective chemical
inducer of apoptosis in human spermatozoa, and has allowed us to
study the biochemical features of this process. Although the range
of apoptotic responses that spermatozoa can express is limited by
their physical architecture, two features appear to be critical. The
first is the expression of annexin V binding as a consequence of
the externalization of PS (Figure 5B). The significance of this
change may be found following insemination when the female
reproductive tract contains millions of moribund and senescent
spermatozoa that must be removed by phagocytic leucocytes.
Accordingly, there is a massive leucocytic infiltration into the
Truncated apoptosis in human spermatozoa
Figure 5
695
Caspase activation and DNA fragmentation following PI3K inhibition
(A) Pan-caspase activation measured by FLICA after a 4 h exposure to wortmannin (***P < 0.001 and *P < 0.05 compared with the vehicle-only control). (B) Time-dependent analysis of caspase
activation and PS externalization demonstrated that the former precedes annexin V binding in the cascade of events that comprise the intrinsic apoptotic response to wortmannin (20 μM). (***P <
0.001 compared with at 0 h) (C) Exposure to common apoptotic stimulants including staurosporine (STS; 10 μg/ml), Kdo (3-deoxy-D-manno-octulosonic acid; 10 μg/ml), lipopolysaccharide (LPS;
10 μg/ml) and genistein (GEN; 10 μM) was unable to induce caspase activation in suspensions of human spermatozoa. (D) The activation of apoptosis with wortmannin did not generate any
detectable DNA damage in human spermatozoa following a 24 h period of exposure, as measured with a TUNEL assay that has been optimized for use with these cells [25].
lower female reproductive tract post coitum [48]. The phagocytic
activity exhibited by these cells must be silent; in other words,
the spermatozoa must be efficiently phagocytosed and removed,
but this activity must not be accompanied by an oxidative burst
or the production of pro-inflammatory cytokines. There are many
examples of silent phagocytosis in biology, and a common feature
of this phenomenon is the expression of apoptotic markers, such as
PS, on the surface of the phagocytosed cell. This apoptotic marker
is thought to instruct the phagocyte that the target cell should be
engulfed in a non-phlogistic manner [49]. We therefore speculate
that the activation of this apoptotic cascade in senescent cells is
an adaptation that permits the efficient removal of spermatozoa
from the female tract by phagocytic leucocytes without provoking
a pro-inflammatory response.
In parallel with the surface expression of annexin V-binding
sites, apoptotic spermatozoa also exhibit signs of caspase
activation (Figure 5A) and a concomitant loss of motility
(Figure 3). Whether there is a causal relationship between
caspase activation and motility loss has not yet been established;
however, these two parameters are highly correlated [50].
Moreover isolation of apoptotic spermatozoa using magnetic
beads coated with annexin V revealed a clear association between
annexin V binding, caspase activation and impaired motility [51].
The motility loss exhibited as part of this apoptotic cascade
could again be an adaptive response to ensure that moribund,
oxidatively stressed spermatozoa with possible DNA damage,
cannot participate in the fertilization process. In most cell
types the activation of the intrinsic apoptotic cascade culminates
in extensive DNA fragmentation and cell death. It is at this point in
the apoptotic pathway that the highly specialized anatomy of
human spermatozoa starts to play a limiting role. Although in
other cell types apoptosis is associated with the movement of
endonucleases (AIFM, CAD, ENDOG) into the nucleus, this
does not, and cannot, occur in spermatozoa. Following the
induction of apoptosis, these proteins [as well as cytochrome
c and DIABLO(SMAC)] remain resolutely locked within the
midpiece region of the cell (Figure 6). Translocation of nucleases
to the sperm nucleus is presumably impeded by the compacted
nature of sperm chromatin and the highly compartmentalized
architecture of mature spermatozoa, which separates the nucleus
in the sperm head from the cytoplasm and mitochondria in the
midpiece. The immediate, practical consequence of endonuclease
exclusion from the sperm nucleus is that these apoptotic cells do
not rapidly exhibit high levels of DNA fragmentation, as measured
in the TUNEL assay (Figure 5D). However, the concomitant
generation of mitochondrial ROS does result in the induction
of oxidative DNA damage, as reflected by 8OHdG formation
(Figure 6K). Since the spontaneous formation of 8OHdG in
human spermatozoa is highly correlated with DNA damage, as
measured with the TUNEL assay [14], it seems probable that
these two events are related and sequential. Thus the formation of
oxidative base adducts will affect DNA integrity by labilizing
the glycosyl bond that attaches the base to the ribose unit,
leading the generation of an abasic site. Abasic sites have a
strong destabilizing effect on the DNA backbone, which can
then result in strand breaks [52]. In light of these considerations
we conclude that, ultimately, apoptosis in human spermatozoa
does result in DNA fragmentation, but it is a prolonged process
driven by oxidative stress rather than endonuclease activity. This
notion does not exclude the possibility that extracellular nucleases
are released by the reproductive tract that dismantle the DNA
of apoptotic spermatozoa post mortem when the integrity of
c The Authors Journal compilation c 2011 Biochemical Society
696
Figure 6
A. J. Koppers and others
Localization of apoptosis mediators and induction of oxidative DNA damage following inhibition of PI3K
Immunocytochemistry revealed that all of the apoptosis mediators examined were confined to the midpiece/principal piece of the spermatozoa prior to the activation of apoptosis (left-hand panels)
and remained fixed in this position 24 h after the induction of apoptosis with 20 μM wortmannin (right-hand panels). (A) and (B) represent CAD, (C) and (D) represent ENDOG, (E) and (F) represent
AIFM, (G) and (H) represent cytochrome c , and (I) and (J) represent DIABLO(SMAC). (K) Although apoptosis in human spermatozoa was not associated with translocation of nucleases to the
sperm nuclei or positive TUNEL signals, the concomitant generation of ROS did result in a significant dose-dependent increase in oxidative damage to the DNA, as measured by 8OHdG formation
(***P < 0.001 and *P < 0.05 compared with the vehicle-only control). Scale bars = 5 μm.
the plasma membrane has been compromised, as suggested by
Dominguez and Ward [53].
Like DNA damage, the changes in mitochondrial activity
observed during apoptosis may also be the result of oxidative
stress. Thus, when this process is triggered by wortmannin or
LY294002, the mitochondria instantly generate ROS, but there
is no immediate loss of . Formation of a mitochondrial
pore without loss of , although uncommon, has been
described previously [54]. In a possible sequence of events,
the dephosphorylation of BAD leads to a loss of cytochrome
c from the mitochondria, which then activates caspases by
participating in apoptosome formation, and alters the activity of
the mitochondrial electron transport chain from the normal fourelectron reduction of O2 to a one-electron process that produces
superoxide anion rather than water [55]. Reverse electron flow to
complex I appears to be responsible for the superoxide generation
observed under these circumstances [56]. This is significant
because we have previously demonstrated that electron leakage
from this site is particularly damaging to spermatozoa, promoting
extensive lipid peroxidation and progressive motility loss [57].
The fact that the remains unchanged during the induction
of mitochondrial ROS generation indicates that depolarization
is not a prerequisite for superoxide production. However, as the
spermatozoa lose vitality, may be secondarily lost as a result
of ROS-induced oxidative damage [58].
c The Authors Journal compilation c 2011 Biochemical Society
In summary, the present study provides definitive evidence
that mature human spermatozoa can be induced to undergo a
limited form of apoptosis characterized by mitochondrial ROS
generation, PS externalization, caspase activation, motility loss,
cytoplasmic vacuole formation and oxidative DNA damage. The
physical architecture of the spermatozoon subsequently prevents
endonucleases released from the mitochondria or activated in the
cytosol from translocating to the nucleus. As a consequence,
DNA fragmentation, one of the hallmarks of apoptosis in
somatic cells, cannot occur immediately, but may be precipitated
secondarily as a result of oxidative DNA adduct formation.
Entry into this apoptotic pathway is actively prevented by
the phosphorylation of PI3K and AKT that, in turn, maintain
BAD in a phosphorylated anti-apoptotic state. Conversely,
dephosphorylation of the PI3K/AKT axis leads to the activation
of apoptosis. We propose that this is an adaptive physiological
response to oxidative stress, rather than a receptor-mediated event.
Furthermore, the purpose of this apoptotic response may be to
both facilitate the silent phagocytosis of senescent moribund
spermatozoa following insemination, and to prevent oxidatively
damaged spermatozoa from participating in the fertilization
process. In light of these findings, we would expect that
growth factors/cytokines that stimulate PI3K/AKT would have a
powerful pro-survival effect on human spermatozoa, as described
recently for prolactin [16]. Furthermore, the active selection of
Truncated apoptosis in human spermatozoa
697
to oxidative stress that can only be counterbalanced by prosurvival factors acting through the PI3K/AKT pathway.
AUTHOR CONTRIBUTION
Adam Koppers and Lisa Mitchell conducted all of the cell biology studies; Ping Wang and
Minjie Lin performed the ultrastructural analysis; and John Aitken designed the study and
prepared the manuscript. All authors contributed to the final version of the paper.
ACKNOWLEDGEMENTS
We thank Jody Powell for orchestrating our semen donor panel.
FUNDING
This work was supported by the ARC Centre of Excellence in Biotechnology and
Development [grant number CEO0348239]; and NSW Department of State and Regional
Development.
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Figure 7 Electron microscopy of human spermatozoa treated with
wortmannin
(A) Untreated morphologically normal spermatozoa. (B and C) Treatment with wortmannin
(20 μM) for 24 h resulted in major morphological changes to these cells, including distension
of the cytoplasm-rich midpiece region of the cell and accompanying vacuole formation, as
indicated by the arrows. Scale bars = 1 μm.
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Although the present studies were conducted on human
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stallion and mouse, in addition to human spermatozoa [59–61].
However, the truncated nature of the apoptotic process in these
cells has not previously been discussed, nor has the concept that
apoptosis represents a default pathway for these cells in response
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