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- Original Article -
Early apoptotic changes in human spermatozoa and their
relationships with conventional semen parameters and
sperm DNA fragmentation
Hao-Bo Zhang1, Shao-Ming
Lu1, Chun-Yan Ma2, Li
Wang1, Xiao Li1, Zi-Jiang
Chen1
1Center for Reproductive Medicine,
2Central Labotrary of Provincial Hospital Affiliated to Shandong University, Jinan
250021, China
Abstract
Aim: To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm
quality.Methods: Two early apoptotic changes in the semen of 56 men were assessed using Annexin V (AN)/propidium
iodide (PI) staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential
(MMP). The results were compared with conventional semen parameters and DNA fragmentation identified using the
TUNEL assay. Results: The different labeling patterns in the bivariate Annexin V/PI analysis identified four
distinctive spermatozoa populations. The percentage of
AN_/PI_ spermatozoa positively correlated with conventional semen
parameters and MMP, but negatively correlated with TUNEL (+) spermatozoa. As for the
AN_/PI+ fraction, we found an opposite result in comparison to
AN_/PI_ spermatozoa. The level of early apoptotic
AN+/PI_ spermatozoa negatively
correlated with MMP and sperm motility. The level of late apoptotic
AN+/PI+ spermatozoa negatively correlated with
conventional semen parameters and MMP, and positively correlated with TUNEL (+) spermatozoa. MMP positively
correlated with conventional semen parameters, but negatively correlated with TUNEL (+)
spermatozoa. Conclusion: Although early apoptotic
AN+/ PI_ spermatozoa only negatively correlates with sperm motility, the differences in
proportion of each subpopulation of spermatozoa (especially, the percentage of
AN_/PI_ spermatozoa), and decreased
MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to
predict the outcome of in vitro fertilization.
(Asian J Androl 2008 Mar; 10: 227_235)
Keywords: Annexin V; apoptosis; DNA fragmentation; infertility; mitochondria; sperm
Correspondence to: Dr Zi-Jiang Chen, Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, 324
Jing-5-Wei-7 Road, Jinan 250021, China.
Tel: +86-531-8518-7856 Fax: +86-531-8706-8226
E-mail: zjchen59@yahoo.com
Received 2006-11-14 Accepted 2007-04-20
DOI: 10.1111/j.1745-7262.2008.00295.x
1 Introduction
Most in vitro fertilization (IVF) cases result from male factor
deficiencies. The quality of sperm is one of the factors determining the success rate of IVF. Currently, sperm quality is evaluated by conventional semen analysis
using a light microscope to determine sperm concentration, motility and morphology. These parameters comply with
World Health Organization criteria [1]. However, conventional semen analysis has limited clinical value for predicting
the success rate of IVF as 50% of couples with failed fertilization have normal pre-IVF semen analysis [2]. Furthermore,
conventional semen analysis does not assess the presence of apoptotic spermatozoa, which might be partially
responsible for the low fertilization and implantation rates in assisted
reproduction. Characteristics that are typical of
ejaculated apoptotic human spermatozoa include phosphatidylserine (PS) externalization, decreased
mitochondrial membrane potential
(MMP), caspase activation and DNA fragmentation [3, 4].
The plasma membrane is a key organelle for sperm
function. Successful fertilization requires a sperm plasma
membrane with normal integrity and function. An early
apoptotic event in somatic cells is characterized by the
loss of phospholipid asymmetry, such as translocation
of PS from the inner to the outer leaflet of the membrane
[3]. Exposure of PS on the outside of the cell surface,
referred to as PS externalization, provides an
opportunity to detect cells that are in the early stage of apoptosis.
In sperm, such cells can be identified by the
calcium-dependent binding of fluorescence-conjugated
Annexin-V (AN) to externalized PS in combination with staining
by fluorescent dyes, such as propidium iodide (PI), which
make it possible to simultaneously distinguish live and
dead spermatozoa. Mitochondria are also a key organelle
for sperm function. In humans, a correlation exists
between poor sperm mitochondrial function detected by
reduced MMP, diminished motility and reduced fertility
[5]. Analysis of mitochondrial function might offer a
means to assess the motility of sperm. Mitochondria are
the coordinators of apoptosis in various cellular systems
because they are involved in many apoptotic processes,
including caspase activation, decreased MMP and
alterations of the intracellular reduction-oxidation potential [6].
PS externalization and decreased MMP are two
characteristics of early stages of apoptosis in somatic cells.
These changes precede other manifestations of programmed cell death, such as DNA fragmentation. TUNEL
assay is commonly used for measuring DNA fragmentation, which is correlated to the outcome of IVF in
humans [7]. The extent of DNA fragmentation is closely
related to sperm function and male infertility [8, 9];
however, the hypothesis about the origin of such
damage is still controversial. The significance of the two
early apoptotic changes encountered in infertile sperm
remains unknown. Hence, we investigated the possible
correlations of the two early apoptotic changes with
conventional semen parameters and DNA fragmentation
identified by TUNEL assay. Our research question is "Can
early apoptotic changes be significant markers of sperm
quality?"
2 Materials and methods
2.1 Semen collection and processing
We studied 56 men who underwent seminal fluid evaluation for various reasons at the Reproductive
Medical Center of Shandong University, Shandong Provincial
Hospital (Jinan, China). All subjects were partners of
women who had failed to conceive after 2 years of
unprotected intercourse. The ethics committee of the
hospital approved the study. Informed consent for
participation in the study was obtained from all participants.
The samples were collected by masturbation into sterile
plastic jars after 3_5 days of sexual abstinence. Within
1 h of collection, routine semen analysis was performed
using a light microscope to determine sperm quality
according to World Health Organization criteria
[10].
2.2 Evaluation of conventional sperm parameters:
concentration, motility and morphology
Sperm concentration, motility and morphology were
evaluated in the original raw sample. After liquefaction,
10 µL of each sample were placed into a Makler
Chamber and read under a light microscope. Sperm
morpho-logy was assessed according to strict criteria at 1 000 ×
magnification. Each sample (10 µL) was spread along
the slide and allowed to dry for 20 min before staining
with Diff-Quik staining. An average of 100 spermatozoa
per slide was read under microscope with a 100 × oil
immersion objective lens. Coefficients of inter-observer
and intra-observer variability were 5%.
2.3 Evaluation of externalization of PS with AN
An AN-fluorescein isothiocyanate (FITC) Apoptosis
Detection Kit (BMS306FI, Bender, Grünberg, Germany)
was used to detect the translocation of PS from the
inner to the outer leaflet of the plasma membrane of
spermatozoa as recommended by the manufacturer with
slight modifications. Generally, an aliquot of semen
specimen containing
1 × 106 spermatozoa were washed
twice (300 × g, 10 min, 4ºC) in phosphate buffered
saline (PBS). The sperm pellet was re-suspended in
ANBinding Buffer (10 mmol/L Hepes/NaOH, pH 7.4,
140 mmol/L NaCl, 2.5 mmol/L
CaCl2) at room temperature to a concentration of
2 × 106 sperm/mL. Aliquots
(100 µL each,
2 × 105 cells) of sperm were transferred
into culture tubes. 5 µL of AN-FITC and 10 µL of PI
(20 µg/mL), or nothing were added to the samples
(nothing was added in the case of the negative
control). Single color staining with either AN-FITC or PI alone
was applied to adjust the compensation. The tubes were
gently mixed and incubated for 15 min at room
temperature in the dark, and additional Binding Buffer (400 µL)
(BMS306FI, Bender, Grünberg, Germany) was added
to each tube. Flow cytometric evaluation was conducted
within 5 min.
2.4 Determination of the integrity of MMP
A lipophilic cationic dye JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenimidazolyl carbocyanine iodide) was
used to detect intact trans-membrane potential of
mitochondria in spermatozoa (MitoCapture Apoptosis
Detection Kit: Catalogue No. 475866; Calbiochem, La Jolla,
CA, USA). Spermatozoa with intact mitochondria excite an intense red fluorescence due to the formation of
the dye aggregates, whereas the monomer dye fluoresces
green in the presence of spermatozoa with a disrupted
mitochondrial membrane. Compensation between FL1 and FL2 was carefully adjusted according to the
manufacturer's instructions. All aliquots were incubated
at 37ºC for 20 min in 1 μg of the lipophilic cation diluted
in 1 mL PBS. Negative controls were identically
processed for each fraction except that the stain was
replaced with 10 µL PBS. Flow cytometric evaluation was
conducted within 30 min.
2.5 TUNEL assay
TUNEL assay in human sperm was conducted in our
laboratory using Fluorescein FragEL DNA Fragmentation
Detection kit (Catalogue No. QIA39; Calbiochem, La Jolla,
CA, USA). Approximately
2 × 106 sperm were first
washed with PBS, and then fixed in 4% paraformaldehyde for 10 min at room temperature. The fixed cells
were washed in TBS (2 mmol/L Tris pH 7.6, 140 mmol/L
NaCl ), and then re-suspended in 20 µg/mL proteinase K
for 10 min at room temperature. After washing with
terminal deoxynucleotidyl transferase (Tdt) equilibration
buffer, the cells were gently re-suspended in Tdt
reaction solution containing the Tdt enzyme and
FITC-labeled nucleotides. For each batch, a negative control
without the addition of Tdt enzyme and a positive
control with DNase I treatment were always included to
ensure the reproducibility of the assay. After incubation in
a humidified chamber for 60 min at 37ºC in the dark, the
sample was analyzed using flow cytometry.
2.7 Flow cytometric analysis
The extent of externalized PS, MMP and TUNEL were
evaluated by flow cytometer analyses. All of the
fluorescence signals of labeled spermatozoa were analyzed
by the flow cytometer (Beckman-Coulter EpicsXL-4; Beckman-Coulter, Inc., Fullerton, CA, USA). A
minimum of 10 000 spermatozoa were examined for each
assay at a flow rate of < 100 cells/s. The sperm
population was gated using 90° 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
FL-1 channel and red fluorescence (580_630 nm) in the
FL-2 channel. The percentage of positive cells and the
mean fluorescence were evaluated on a 1023-channel
scale using the flow cytometer System II Version 3.0
software (Beckmann Coulter, Miami, FL, USA).
2.8 Statistical analysis
Data are presented as mean ± SD. Results were
analyzed using SPSS version 10.0 software for Windows
(SPSS, Chicago, IL, USA). The Pearson rank correlation test was used to calculate the correlation coefficient
between cytofluorometric analyses. The Spearman rank
correlation test was used to evaluate the relationship
between conventional semen parameters and
cytofluorometric examination. The statistical significance level was
set at P < 0.05.
3 Results
A total of 56 men who visited the reproductive
medical center during the study period (April 2006_July 2006)
were recruited. Their ages ranged from 24 to
42 years (31 ± 4 years). The results of the routine semen
analyses are shown in Table 1. The semen samples had a
sperm concentration of (42 ± 30) ×
106/mL, 48% ± 15% of the spermatozoa appeared to be motile, and 29%
± 3% of the spermatozoa had normal forms.
In the present study, three assays were applied to
examine the apoptotic changes in ejaculated sperm (Figures 1_3). These assays are based on flow cytometry
and are more accurate, rapid and can provide less
subjective and statistically more reliable results than
microscopic examination. The different labeling patterns in
the bivariate AN/PI analysis identified four distinctive
spermatozoa populations: live spermatozoa with no
translocation of membrane PS, namely
`AN-/ PI-'; live spermatozoa with translocation of membrane
PS, namely `AN+/ PI-'; dead spermatozoa with translocation of membrane
PS, namely `AN+/PI+'; and dead spermatozoa with no
binding of AN, namely `AN-/
PI+'. The distributions of the populations are as follows:
AN_/PI_, (55 ± 16)%;
AN+/PI_, (10 ± 6)%;
AN+/PI+, (23 ± 10)%;
AN_/PI+, (11 ± 7)%.
The MMP and DNA fragmentation (TUNEL assay) of the
semen samples are as follows: (55 ± 22)% and
(23 ±14)%, respectively (Table 2).
To assess the possible impact of sperm apoptosis on
sperm quality, we further analyzed the correlations
between the apoptotic indices and various semen parameters, and the results are summarized in
Table 3. We found that the amounts of both
AN+/PI+ (late apoptosis) spermatozoa and TUNEL (+) spermatozoa
were inversely correlated to concentration, motility and
sperm normal morphology. In contrast, no correlation
was observed between the amount of
AN+/PI_ (early apoptosis) spermatozoa and sperm concentration or
sperm normal morphology. However, the amount of
AN+/PI_ (early apoptosis) spermatozoa is inversely
correlated with motility. We also found that MMP and the
amount of AN_/PI_ spermatozoa positively correlated with
concentration, motility and sperm normal morphology.
The correlations of the two early apoptotic alterations
with the TUNEL assay are summarized in Table 4. The
amount of TUNEL (+) spermatozoa positively correlated
with the levels of both
AN+/PI+ (late apoptosis) and
AN_/PI+ spermatozoa, but negatively correlated with both the level
of AN_/PI_ spermatozoa and MMP. In contrast, MMP
positively correlated with the level of
AN_/PI_ spermatozoa, but negatively correlated with the levels of
AN+/PI+ (late apoptosis),
AN+/PI_ (early apoptosis) and
AN_/PI+ spermatozoa.
4 Discussion
Apoptosis is an important process involved in normal
spermatogenesis [11]. However, deregulations of this
biological process involve abnormalities in the production of
male gametes, and male infertility. In recent years, much
attention has been given to the role of apoptosis in
ejaculated sperm. To date, however, whether defective apoptosis
accounts for a significant proportion of DNA damage in the
spermatozoa of infertile men is still questionable. In this
regard, we hypothesize that the differences in proportion
of each subpopulation of spermatozoa (according to the
AN/PI staining) and decreased MMP of the semen sample
might correlate with the quality of sperm.
4.1 AN_/PI_ and
AN_/PI+ spermatozoa
The percentage of AN_/PI_ spermatozoa positively
correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL (+).
AN_/PI_ spermatozoa displayed superior quality in terms of
high motility, low activated caspases, high MMP and
small extent of DNA fragmentation [12, 13]. Moreover,
AN_/PI_ spermatozoa might be a sperm subpopulation
of good functional competence that is able to adapt to
freeze/thaw-induced stress. These findings lead us to
postulate that the AN_/PI_ spermatozoa represent high
quality sperm so as to serve as an objective indicator for
the fertility potential of an individual. As for the
AN_/PI+ fraction, we found an opposite result in comparison to
AN_/PI_ spermatozoa. Such spermatozoa exist, but have
not been described by some of other researches [14,
15]. It has been suggested these spermatozoa are
either necrotic cells characterized by a high degree of
membrane disorganization, which might prevent binding of AN, or spermatozoa in the later stage of apoptosis
[16]. However, the significance of this subpopulation
needs to be investigated further.
4.2 AN+/PI_ and
AN+/PI+ spermatozoa
Controversy exists as to the correlation of the amount
of AN+/PI_ group with motility, i.e. some studies report
negative correlations [13, 16] or a positive correlation
[3], whereas other reports no correlation [16]. Our study
demonstrated that the levels of both early apoptotic
AN+/PI_ spermatozoa and late apoptotic
AN+/PI+ spermatozoa negatively correlated with sperm motility. There are two
major hypotheses explaining the externalization of PS as
visualized by AN-staining. It might be an early event in
spermatozoa apoptosis and/or a reflection of sperm
capacitation. Capacitation is associated with an increase
in the motion parameters, wheras motility is reduced in
apoptotic sperm. Our results support the apoptosis
hypothesis. Moreover, we demonstrated that
the late apoptotic alterations in sperm were positively associated
with abnormal sperm morphology. Our results are, at
least in part, consistent with previous findings
[15]. The results can be explained by the abortive apoptosis theory
proposed by Sakkas et al. [8, 17]. With respect to the
relationship between apoptotic changes and the sperm
concentration, available data are still controversial.
Oosterhuis et al. [18] found that the level of
spermatozoa that express PS (AN+) or the percent of TUNEL (+)
sperm negatively correlated with sperm concentration.
In contrast, Shen et al
. [15] found that late apoptotic alterations in sperm positively correlated with sperm
concentration. Our results contradict those of
Shen et al. [15] and can also be explained by the abortive apoptosis
theory. One possible explanation for the finding of
Shen et al. [15] is that the sperm samples used in their study
had two distinctive features: (i) most of them were within
the normal range of sperm concentration; and (ii) the
percentage of sperm defects was unusually high. Additionally, we found that the percentage of early
apoptotic AN+/PI_ spermatozoa was not correlated with
the percentage of TUNEL (+) spermatozoa but the
percentage of late apoptotic AN+/
PI+ spermatozoa was positively correlated with the percentage of TUNEL (+)
spermatozoa. This can be explained by the common understanding that DNA fragmentation usually occurs at
the late stage of apoptosis caused by endonuclease
activation. Such correlations also suggest that the
AN+/ PI+ spermatozoa are more likely to be late apoptotic cells,
rather than necrotic cells. These results pointed to an
abortive apoptosis that takes place in many men who
have sperm parameters that are below normal. Apoptosis
in mature sperm is initiated during spermatogenesis in
which some cells, earmarked for elimination, might
escape the removal mechanism and contribute to poor sperm
quality [15, 19]. In certain men, abortive apoptosis might
fail in the total clearance of spermatozoa earmarked for
elimination by apoptosis. In our study, significant
correlations are reported between apoptotic changes
(according to the AN/PI staining) in sperm with some
conventional sperm parameters (Table 3). However, such
results appear in contrast with previous similar works
[16]. This difference could be a result of the different
method used and/or to the different patient population
studied.
4.3 MMP versus conventional sperm parameters and
apoptotic changes
At present, MMP is considered a good indicator of
sperm motility. Our results, together with other observations, indicates that mitochondrial damage might
result in a reduction of sperm motility [5, 19]. It might
also provide a possible explanation for poor sperm
motility in asthenozoospermic samples. In addition, we found
that MMP positively correlated with sperm
concentration and sperm morphology. This result is in line with
the data reported by other researchers [5, 20]. Marcheeti
et al. [21] found that determination of MMP represents
the most sensitive test by which to evaluate sperm quality.
Furthermore, our study also showed that MMP negatively correlated with the percentage of TUNEL (+)
spermatozoa. This finding strongly implied an
association of male infertility with mitochondrial alterations
observed during apoptosis. However, it is not known if
apoptosis hypotheses could explain the overall DNA
damage or if there are others factors involved in the final
DNA damage. Sakkas et al. [22] showed that DNA
fragmentation is not always concomitant with the
expression of the apoptotic markers (such as Fas, p53 and
Bcl-x) and that problems in nuclear remodeling of
spermiogenesis might account for a fraction of DNA
fragmentation. Wang et al. [20] reported that increased
oxidative stress is associated with alterations in MMP
and increased spermatozoa DNA damage. This can also
explain the negative relationship of our result. In addition,
Moustafa et al. [23] observe a strong correlation
between reactive oxygen species (ROS) and the level of
apoptosis. This might reflect a causal relationship
between ROS and apoptosis. As mentioned above, the
significance of the decreased MMP encountered in infertile
sperm remains unknown but the assumption that it
manifests an early stage of cell death might explain why
detection of mitochondrial changes represents a sensitive
test in our study.
In conclusion, although early apoptotic
AN+/PI_spermatozoa only negatively correlates with sperm
motility, the differences in proportion of each
subpopulation of spermatozoa (especially the percentage of
AN_/PI_ spermatozoa), and decreased MMP might be
significant markers for diagnosing male infertility. They
possibly bring additional information to predict the
outcome of IVF. Further studies are needed to determine
their relationships with the occurrence of pregnancy or
fertilization in vitro.
Acknowledgment
This work was supported by grants from the National Natural Science Foundation of China (No.
30470703). The authors would like to thank Dr
Jian-Feng Li for his valuable comments and
assistance.
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