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- Original Article -
Stability of fluorochrome based assays to measure subcellular
Sonja Grunewald, Manja Rasch, Martin Reinhardt, Thomas Baumann, Uwe Paasch, Hans-Juergen Glander
Department of Dermatology/Andrology Unit, European Academy of Andrology (EAA) Training Center, University of Leipzig,
Leipzig 04103, Germany
Aim: To evaluate the long-term stability of the fluorescence signals of new fluorescence-based semen analysis assays
for clinical application. Methods: Semen samples from 87 unselected infertile patients were used to perform the
following assays: (i) detection of active caspase-3
(n = 17); (ii) integrity of the mitochondrial membrane potential
(MMP) (n = 17); (iii) externalization of phosphatidylserine (EPS)
(n = 16); and (iv) detection of intact acrosomes via
CD46 (n = 37). After the assays, 4% paraformaldehyde was added to all aliquots. The fluorescence intensity of each
sample was evaluated by flow cytometry on days 0, 3, 7, 10 and
14. Results: Differences of up to ± 5% positive
spermatozoa from the value measured at day 0 were estimated as acceptable deviation. The Caspase-3
FLICATM showed mean differences < 5% at day 3, 7 and 10. At day 14 the mean difference was 7.6%. In contrast, the
disrupted MMP and the EPS detection showed differences > 5% at day 3. The CD46-FITC labeling displayed
absolute differences < 5% CD46-positive spermatozoa at days 3, 7, 10 and
14. Conclusion: Although immediate analysis
of the fluorescence signals is recommended, it is possible to evaluate caspase-3 activation up to 10 days and CD46 up
to 14 days after staining of sperm. The FACS evaluation of MMP and EPS detection should be conducted on the same
day. (Asian J Androl 2008 May; 10: 455_459)
Keywords: human spermatozoa; apoptosis assays; caspase-3; mitochondrial membrane potential integrity; CD46; semen analysis
Correspondence to: Dr Hans-Juergen Glander, Department of Dermatology/Andrology Unit, European Academy of Andrology (EAA)
Training Center, University of Leipzig, Ph.-Rosenthal-Str. 23-25, Leipzig 04103, Germany.
Tel: +49-3419-718-740 Fax: +49-3419-718-749
Received 2007-10-11 Accepted 2008-02-20
Applications of assisted reproductive techniques (ART) to treat infertile couples have expanded rapidly within the
past decade. However, the pathophysiological diagnosis of male infertility is often missed and the current pregnancy
and live-births success rates remain unsatisfactory . Standard semen analysis does not provide information about
impaired sub-cellular processes in human sperm . Several fluorescence-based techniques have been applied to
evaluate functional ultrastructure of sperm defects: the integrity of the plasma membrane,
the mitochondrial transmembrane potential (MMP) and caspase-3 activation
[3_5]. These variables, which represent components of the apoptosis
signaling cascade from somatic cells  correlate well with sperm motility
[7_9] and morphology  as well as with the fertilizing ability of human sperm [11, 12]. Sperm preparation techniques used before ART procedures, like
cryopreservation and thawing or centrifugation, induce sub-cellular damage in various segments of sperm . An
increase in the numbers of sperm having externalized phosphatidylserine in the outer plasma membrane, showing a
disrupted MMP, or possessing activated caspase-3 (cytosolic aspartate-specific protease 3) might play a role in male
infertility , and their depletion might improve the
outcome of ART.
The measurement of the percentage of acrosome-reacted sperm before and after induction of the acrosome
reaction gives insight as to the functional ultrastructure
of sperm. Decreased proportions of acrosome-reacted
sperm after induction of the acrosome reaction are found
significantly more often in ejaculates from subfertile
patients . Labeling with anti-CD46 antibodies is an
established method to measure the amount of acrosome-
reacted sperm [16, 17]. The integrity of the MMP and
the activation of caspase-3 can be evaluated using
commercially available kits. FITC-labeled antibodies raised
against phosphatidylserine and CD46 are used to detect
membrane changes and the proportion of acrosome-reacted sperm, respectively.
All of the previously mentioned assays are
fluorescence-based. The evaluation by fluorescence
microscopy reveals much more imprecise results compared to
flow cytometric analysis. Fluorescence microscopy
allows the measurement of only a limited number of cells
because of fading under the fluorescence light and due
to time limitations. For example, a recent study
demonstrated in comparison to flow cytometric analysis
consistently lower levels of caspase-activation when the
evaluation was performed by fluorescence microscopy.
However, the intra-observer and inter-observer
reliability of both methods were comparable . Immediate
access to a flow cytometer is not possible in all laboratories.
The present study was conducted to evaluate the
stability of the fluorescence signals of these recently
developed tests for clinical application.
2 Materials and methods
2.1 Samples and experimental design
This study was approved by the Institution Review
Board of the Faculty of Medicine, University of Leipzig
(Leipzig, Germany). Aliquots were collected from
semen samples of 87 unselected male partners of infertile
couples after given informed consent. Following
liquefaction and density gradient centrifugation at 500 ×
g for 20 min (PureCeption, SAGE BioPharma, Bedminster, NJ,
USA), the resulting 80% pellet was re-suspended in
phosphate buffered saline (PBS: pH 7.4,
Na2HPO4 8.0 mmol/L,
K2HPO4 1.5 mmol/L, KCl 2.7 mmol/L, NaCl
140.0 mmol/L) for the various assays.
2.2 Monitoring of externalization of phosphatidylserine
EPS was examined in 16 semen samples using a monoclonal mouse anti-human phosphatidylserine
antibody, clone 1H6 (Upstate cell signaling solutions, Lake
Placid, NY, USA). Spermatozoa were incubated with
the phosphatidylserine-antibody at a final concentration
of 0.5 µg/mL in PBSB (PBS, pH 7.4 containing 2%
bovine serum albumin [BSA]) for 20 min on ice, followed
by addition of 150 µL PBSB and centrifugation at 400 ×
g for 5 min at 20ºC. After discarding the supernatant each
sperm pellet was incubated protected from light with
50 µL of secondary antibody (goat anti-mouse IgG [H +
L], fluorescein conjugate, Upstate Cell Signalling
Solutions) on ice for 20 min. A second washing step in
PBSB (400 × g for 5 min at 20ºC) was performed to
remove excess antibody that was not bound to the
spermatozoal surface. For assessment by flow cytometry,
sperm pellets were diluted in 400 µL PBSB. Human
neutrophils (5 × 106 cells) treated with 1 mmol/L
cycloheximide for 6 h served as positive controls for induction of
apoptosis. The negative controls were processed
identically for each fraction, except that the primary antibody
was replaced with 200 µL PBS.
2.3 Detection of activated caspase-3
In 17 aliquots, the levels of activated caspase-3 were
detected using a fluorescein-labeled inhibitor of
caspase-3, which is cell permeable, non-cytotoxic, and binds
covalently to activated caspase-3 . The inhibitor was
used with the appropriate controls according to the kit
instructions provided by the manufacturers (Fluorescence
Labeled Inhibitor of Caspase-3, Caspase-3
FLICATM; Immunochemistry Technologies, Bloomington, MN, USA).
A 150-fold stock solution of the inhibitor was prepared
by dissolving the lyophilized caspase-inhibitor in 50 µL
dimethyl sulfoxide (DMSO) and was further diluted 1:5
in PBS to yield a 30-fold working solution (per aliquot:
2 µL of the stock solution plus 8 µL PBS). All test
aliquots and controls (with 100 µL PBS) were incubated
at 37ºC for 1 h with 10 µL of the working solution and
subsequently washed with the rinse buffer. In
concordance with the monitoring of MMP, human neutrophils
(5 × 106 cells) treated with 1 mmol/L cycloheximide for
6 h were used as positive controls for induction of
apoptosis. The negative controls were processed
identically for each fraction, except that the stain was replaced
with 10 µL PBS.
2.4 Monitoring of MMP
In further 17 aliquots a lipophilic cationic dye was
applied. Sperm with intact mitochondria emit an intense
red fluorescence as a result of the formation of dye
aggregates. The monomer dye fluoresces green, indicating a
disrupted MMP. MitosensorTM was used according to
the instructions of the manufacturer (Apoalert
Mitochondrial Membrane Sensor Kit; Clontec Laboratories, Palo
Alto, CA, USA). Briefly, all aliquots were incubated at
37ºC for 20 min in 1 µg of the lipophilic cation diluted in
1 mL PBS. Human neutrophils
(5 × 106 cells) treated with 1 mmol/L cycloheximide for 6 h were used as
positive controls for induction of apoptosis. Negative
controls were processed identically for each fraction except
that the stain was replaced with 1 mL PBS.
2.5 Evaluation of the acrosomal status
In 37 aliquots, the acrosome reaction was monitored
with and without induction. Sperm were capacitated for
3 h at 37ºC with 5% CO2 in human tubar fluid (HTF)
medium containing 3 mg/mL BSA (Purity = 98%, Merck;
Darmstadt, Germany). Induction of acrosome reaction
was performed using the calcium ionophore A23187 (Sigma; St. Louis, MO, USA). On the day of use, a
frozen aliquot of stock solution of calcium ionophore
A23187 in DMSO (Sigma) was diluted 1:5 with HTF medium and 20 µL of the solution was added to 100 µL
of the sperm suspension. As a control, an aliquot of the
same sperm suspension was left untreated. Both tubes
were incubated for 1 h at 37ºC in 5%
CO2 in air before the acrosomes were assessed.
The amount of spontaneous and ionophore-induced
acrosome reaction was detected using a monoclonal FITC-labeled mouse anti-human CD46 antibody (IgG2a;
Biomeda, Foster City, CA, USA). All aliquots were
washed in 900 µL PBS for 4 min at 400 ×
g. The resulting sperm pellet was resuspended in
100 μL PBS containing 4 μL of CD46-FITC and incubated for 30 min
under light protection. A second washing step in PBS
(4 min at 400 × g) was performed before the pellet was
resuspended in 0.4 mL PBS.
2.6 Flow cytometry analysis
The extent of CD46 and EPS on the sperm surface
as well as the percentage of sperm with activated
caspase-3 and a disrupted MMP was evaluated by flow cytometry
in neat aliquots at day 0 and after the addition of 4%
paraformaldehyde 1:1 to the remaining portion of all
samples on days 0, 3, 7, 10 and 14. A minimum of
10 000 spermatozoa was examined for each assay at a
flow rate of < 100 cells/s. The sperm population was
gated using 90 degree and forward-angle light scatter to
exclude debris and aggregates. The excitation
wavelength was 488 nm, supplied by an argon laser at 15 mW.
Green fluorescence (480_530 nm) was measured in the
FHL-1 channel and red fluorescence (580_630nm) in the
FHL-2 channel. The percentage of positive cells and the
mean fluorescence was calculated on a 1023 channel
scale using software Expo32ADC (Coulter, Krefeld, Germany).
2.7 Statistical analysis
Data were analyzed using inbuilt functions within the
Statistica 6.0 software (StatSoft; Tulsa, OK, USA). Study
variables were not normally distributed. Summary
statistics are presented as mean absolute difference.
Univariate comparison of sperm variables at different time points
was performed with Wilcoxon's signed rank test. All
hypothesis tests were two-tailed. P < 0.05 was
considered statistically significant.
All applied assays can be performed in a reasonable
timeframe: for measurement of the integrity of the MMP
only 30 min is necessary, for detection of the amount of
caspase-3 positive sperm 70 min is required, and for the
evaluation of EPS 60 min is needed. To label sperm with
the anti-CD46-FITC antibody, 40 min is required, but
prior capacitation takes 3 h. Depending on the number
of samples it is possible to run the assays simultaneously.
The percentage of sperm containing activated
caspase-3, an intact or disrupted MMP, EPS and CD46 was
detected by FACS analysis on days 0, 3, 7, 10 and 14.
The fluorescence signals of all assays diminished with time,
but there were both positive and negative differences when
compared to day 0. The differences (Δx) between the
values measured at day 0 (x0) and day 3
(x1), day 7 (x2), day 10
(x3) and day 14 (x4) were
evaluated as absolute values of differences: Δx =
|xi _ xj| (i, j =
0_4). Absolute differences of up to 5% of positive sperm from the value
measured at day 0 were estimated as an acceptable
Caspase-3 activation was detected in 55% ± 18% of
spermatozoa on day 0 (mean ± SD). The FACS analysis
showed mean differences < 5% on day 3
(3.7%), day 7 (3.5%) and day 10 (3.2%). The mean absolute difference
on day 14 was 7.6% (Figure 1A). The
MitosensorTM revealed that 41% ± 24% of the sperm contained a
disrupted MMP on day 0. In contrast to the Caspase-3
FLICATM the fluorescence signal was not stable,
showing mean differences of 6% (day 3), 31% (day 7), 17%
(day 10) and 34% (day 14) of sperm with a disrupted
MMP (Figure 1B). Similar results were derived from
stability analyses of EPS detection: 42% ± 22% of sperm
were EPS positive at day 0. Flow cytometry of the same
aliquots demonstrated mean differences of 7.8% (day 3),
13% (day 7), 12% (day 10) and 17% on day 14 (Figure 1C).
CD46-FITC labeling (before induction of acrosome reaction: 6.9% ± 12%, after induction of acrosome
reaction: 44% ± 16% CD46 positive sperm) displayed
absolute differences < 5% of CD46 positive
sperm at days 3 (1.5%), 7 (2.9%), 10 (3.8%) and 14 (4.5%,
Ejaculates of infertility patients often demonstrate
normal sperm variables as determined by standard
semen analysis . Results derived from
fluorescence-based assays like Caspase-3
FLICTM, MitosensorTM or the FITC-based EPS and CD46 detection will provide a
better diagnosis in the future. There is significant
increase in caspase activation in various cases of
"unexplained" infertility . In addition, measurement of
subcellular markers like apoptosis signaling allows
further optimization of sperm preparation (e.g. during
cryopreservation and thawing) . The novel assays
will increase our knowledge of the pathophysiology of
known conditions like varicocele associated with an
increase in caspase activation . Methods widely used
in ART like centrifugation, cryopreservation and
thawing might be subjected to further optimization according
to cellular changes on a molecular level . The
commercially available assays as well as the detection by
FITC-coupled antibodies are easy to perform in a
reasonable timeframe. The Caspase-3
FLICATM showed mean differences of < 5% until day 10. In contrast, the
MitosensorTM and the EPS detection showed differences
of > 5% as early as day 3. The CD46-FITC labeling
displayed absolute differences of < 5% of CD46 positive
sperm on days 3, 7, 10 and 14. Although immediate
analysis of the fluorescence signals is recommended, it
is possible to evaluate activation of caspase-3 up to
10 days after staining of human sperm. Labeling with
anti-CD46-FITC on the sperm surface gives very stable
fluorescence signals and flow cytometric analysis can
be performed within at least the following 2 weeks. The
FACS evaluation of MMP integrity and EPS detection
should be conducted on the same day.
We would be grateful to funds from the German
Research Council (Deutsche Forschungsgemeinschaft, DFG
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