1 Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute, and 3 Department of Biostatistics and Epidemiology, The Cleveland Clinic Foundation, Cleveland, OH, USA and 2 Cairo University, Cairo, Egypt
4 To whom correspondence should be addressed at: Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute and Department of ObstetricsGynecology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Desk A19.1, Cleveland, OH 44195, USA. e-mail: agarwaa{at}ccf.org
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Abstract |
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Key words: apoptosis/DNA damage/reactive oxygen species/spermatozoa
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Introduction |
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Alterations in the spermatogenic events result in
the release of immature, abnormal spermatozoa in the ejaculate. Immature
spermatozoa display a high content of DNA damage, alterations in
protamination, chromatin packaging and excessive reactive oxygen species
(ROS) production (Balhorn et
al., 1988; Gorczyca et al., 1993a
,b; Sailer et al., 1995
;
Bianchi et al.,
1996
; de Yebra et
al., 1998
; Manicardi et al., 1998
; Evenson et al., 2000
).
Spermatozoa from infertile men have been shown to contain various nuclear
alterations, including abnormal chromatin structure, microdeletions,
chromosomal rearrangements, aneuploidy and DNA strand breaks (Gerardo et al.,
2000
).
Although the extent of DNA damage is closely
related to sperm function and male infertility (Aitken, 1999; Sakkas et al., 1999
), the origin of
such damage is still largely controversial. Apoptosis is a mode of cellular
death based on a genetic mechanism that induces a series of cellular
morphological and biochemical alterations leading the cell to suicide
(Kerr et al.,
1972
; Nagata,
1997
). There are a number of indications that apoptosis occurs
during spermatogenesis in humans (Gorczyka et al., 1993a
,b; Manicardi et al., 1995
;
Baccetti et al.,
1996
; Hadziselimovic et al., 1997
;
Sun et al.,
1997
; Lopes et
al., 1998
; Manicardi et al., 1998
; Sakkas et al., 1999
;
Barroso et al.,
2000
; Irvine et
al., 2000
). Recently, attention has focused on the role of
apoptosis in ejaculated sperm (Koopman et al., 1994
; Glander and Schaller, 1999
;
Barroso et al.,
2000
; Oosterhuis et
al., 2000
; Duru et al., 2001a
,b; Schuffner et al., 2001
,
2002
; Shen et al., 2002
).
Whether defective apoptosis accounts for a significant proportion of DNA
damage seen in the spermatozoa of infertile men is still an open question.
The observation that mature ejaculated spermatozoa are positive for the
TUNEL assay has led to the theory that apoptosis is occurring (Gorczyca et al., 1993a
,b;
Baccetti et al.,
1996
; Sun et
al., 1997
; Lopes et al., 1998
; Manicardi et al., 1998
;
Tesarik et al.,
1998
).
Another mechanism that has been studied
extensively is oxidative stress, which is caused by the overproduction of
ROS (Sharma and Agarwal,
1996; Aitken and Krausz,
2001
; Sikka,
2001
; Agarwal and Saleh,
2002
; Saleh and
Agarwal, 2002
; Saleh et al., 2002a
,b; Agarwal et al., 2003
). ROS
cause lipid peroxidation of sperm plasma membranes, resulting in alteration
of sperm function and fertilizing capacity (Duru et al., 2000
). They are also
known to affect the sperm genome, causing high frequencies of single-
and double-strand DNA breaks (Twigg et al., 1998
). Both superoxide
(O2.) and the hydroxyl radical (OH·)
are known to be mutagenic and cause chromosome deletions, dicentrics and
sister chromatid exchanges (Twigg et al., 1998
; Aitken and Krausz, 2001
). These
abnormalities in chromatin packaging and nuclear DNA damage appear to be
linked, and there is a strong association between the presence of nuclear
DNA damage in the mature spermatozoa of men and poor semen parameters
(Lopes et al.,
1998
; Irvine et
al., 2000
).
Studies have shown DNA fragmentation in
ejaculated spermatozoa (Aitkenet al., 1998; Irvine et al., 2000
; Zini et al., 2001
;
Saleh et al.,
2002a
). DNA damage has been linked to poor pregnancy outcome
(Sun et al.,
1997
; Lopes et
al., 1998
; Evenson et al., 1999
, 2000
; Host et al., 2000a
,b
; Larson et al., 2000
; Scholl and Stein, 2001
; Duran et al., 2002
;
Benchaib et al.,
2003
; Saleh et
al., 2003
). Three hypotheses have been postulated to
explain the source of DNA damage in sperm. First, it is believed that DNA
damage is caused by improper packaging and ligation during sperm maturation
(McPherson and Longo,
1992
, 1993
a,b; Gorczyca et al., 1993a
,b; Sailer et al., 1995
).
Secondly, oxidative stress causes DNA damage (Agarwal and Saleh, 2002
; Saleh et al., 2002a
,b; Agarwal et al., 2003
), and
the increased levels of specific forms of oxidative damage such as
8-hydroxydeoxyguanosine in sperm DNA supports such a theory
(Lopes et al.,
1998
; Aitken,
1999
; Shen and Ong,
2000
). Thirdly, observed DNA fragmentation is caused by
apoptosis (Sakkas et al.,
1999
, 2002
).
The objectives of our study were (i) to assess the role of apoptosis in the pathogenesis of DNA damage in ejaculated spermatozoa from patients examined for infertility; and (ii) to assess the correlation of apoptosis with conventional semen parameters (sperm concentration, motility and morphology) and ROS levels in sperm from patients examined for infertility.
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Materials and methods |
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Measurement of sperm morphology
Smears of raw semen were prepared for sperm morphology
assessment. The smears were fixed and stained using the Diff-Quik kit
(Baxter Healthcare Corporation, Inc., McGaw, IL). Immediately after
staining, the smears were rinsed in distilled water and air-dried.
Smears were scored for sperm morphology using WHO guidelines (World Health Organization, 1999),
and Tygerberg strict criteria (Mortimer and Menkveld,
2001
).
Isolation of mature sperm population
Aliquots of 0.51 ml of the liquefied semen
were loaded onto a 47 and 90% discontinuous ISolate gradient (Irvine
Scientific, Santa Ana, CA) and centrifuged at 500 g for
20 min at room temperature. The presence of mature and immature
spermatozoa in the two fractions was verified by preparing smears for sperm
morphological abnormalities and the presence of excessive residual
cytoplasm as described in our earlier publication (Gil-Guzman et al., 2001). The
resulting interfaces between the 47% pellet (fraction 1, immature
spermatozoa) and the 90% pellet (fraction 2, containing mature
spermatozoa) were aspirated, and these were transferred to separate test
tubes. The pellet from fraction 2 was suspended in 1 ml of
BiggersWhittenWhittingham medium (BWW) and centrifuged at
500 g for 7 min. The pellet was then re-suspended
in 1 ml of BWW, and an aliquot was examined for sperm concentration,
percentage motility, total sperm count, sperm morphology and ROS
production.
Measurement of reactive oxygen species
Production of ROS was measured in washed semen by the
chemiluminescence assay method using luminol (5-amino-2,
3-dihydro-1, 4-phthalazinedione; Sigma Chemical Co., St
Louis, MO) as a probe (Kobayashi et al., 2001). Aliquots of
liquefied semen were centrifuged at 300 g for 7 min. Seminal
plasma was separated and frozen at 80°C for later measurement of
the total antioxidant capacity (TAC) level. The sperm pellet was washed
twice with phosphate-buffered saline (PBS) pH 7.4, and
resuspended in the same medium at a concentration of
20 x 106 sperm/ml. A 10 µl aliquot of
luminol prepared as 5 mmol/l stock in dimethylsulfoxide (DMSO) was
added to 400 µl of the washed sperm suspension; 10 ml of
5 mol/l luminol added to 400 µl of PBS served as a negative
control. Levels of ROS were determined by measuring chemiluminescence with
a luminometer (LKB 953, Wallac Inc., Gaithursburg, MD) in the integrated
mode for 15 min. Results were expressed in
x106 c.p.m. per 20 x 106
sperm per ml.
Total non-enzymatic antioxidant capacity was
measured in the seminal plasma with an enhanced chemiluminescence assay
(Saleh et al.,
2002b). Liquefied semen samples were centrifuged at
250 g for 7 min, and seminal plasma was separated and
stored at 80°C. Frozen samples of seminal plasma were thawed at
room temperature, diluted 1:20 with de-ionized water (dH2O)
and filtered through a 0.20 µl filter (Allegiance Healthcare
Corporation, McGaw Park, IL). Signal reagent was prepared by adding
30 µl of H2O2 (8.8 molar/l),
10 µl of para-iodophenol stock solution
(41.72 µmol/l) and 110 µl of luminol stock solution
(3.1 mol/l) to 10 ml of Tris buffer (0.1 mol/l,
pH 8.0). Horseradish peroxidase (HRP) working solution was prepared
from HRP stock solution by making a dilution of 1:1 of dH2O to
give a chemiluminescence output of
3 x 107 c.p.m. Trolox
(6-hydroxyl-2,5,8-tetramethylchroman-2-carboxylic
acid), a water-soluble tocopherol analogue, was prepared as a standard
solution (25, 50 and 75 µmol/l) for TAC calibration.
With the luminometer in the kinetic mode, 100 µl of signal reagent and 100 µl of HRP working solution were added to 700 µl of dH2O and mixed. The mixture was equilibrated to the desired level of chemiluminescence output (between 2.8 and 3.2 x 107 c.p.m.) for 100 s. Three concentrations (25, 50 and 75 µmol/l) of 100 mmol/l of standard Trolox solution were immediately added to the mixture, and chemiluminescence was measured. Suppression of luminescence and the time from the addition of seminal plasma to 10% recovery of the initial chemiluminescence were recorded. The same steps were repeated with replacement of Trolox solutions with 100 µl aliquots of the prepared seminal plasma.
TAC calculation
Seminal
TAC levels were calculated using the following
equation:
Y = (Mx ± C) x d.
In this equation, Y = antioxidant concentration in µM; M = slope of the curve; x = recovery time in seconds (the shorter the recovery time, the lower the amount of antioxidant present in the sample); C = intercept and not the daily variability (this will be zero if the intercept passes through zero, positive if the intercept passes above zero, or negative if it passes below zero); and d = dilution factor and expressed as molar Trolox equivalents.
Assessment of apoptosis by annexin-V staining
Externalization of phosphatidylserine (PS) to the outer
leaflet of the plasma membrane is an early step in the apoptotic process.
Annexin V is a calcium-dependent phospholipid-binding protein
with a very high affinity for PS. Annexin-V binding was examined in
aliquot I and in fractions 1 and 2 (Vermes et al., 1995). Once the
annexin-V label had been applied, spermatozoa were assessed using
epifluorescent microscopy. To differentiate apoptotic from necrotic
spermatozoa, we included propidium iodide (PI) stain.
Exclusion of PI coupled with binding of annexin-V indicates membrane changes that are characteristic of early apoptosis. Complete disruption of the plasma membrane as seen in necrosis allows for both PS and PI to be expressed. Samples were classified as normal (negative annexin and PI), apoptotic (positive annexin-V and negative PI) or necrotic (positive PI). From both sperm fractions, sperm cells were resuspended in PBS (Sigma) and incubated with annexin-V reagent in HEPES buffer containing PI. The sample was analysed using epifluorescent microscopy. An excitation wavelength in the range of 450500 nm and a detection wavelength in the range of 515565 nm were used. A total of 200 spermatozoa were randomly assessed per slide in five fields and identified as normal, apoptotic or necrotic.
Measurement of mature sperm nuclear DNA damage by SCSA
DNA damage was measured in separate
aliquots after a simple wash and density gradient centrifugation (fractions
1 and 2) by sperm chromatin structure assay (SCSA; Evenson et al., 1999, 2002
). A total of 5000 acridine
orange-stained sperm were measured at a rate of
250 cells/s for
the amount of green (515530 nm = native DNA)
and red (> 630 nm = denatured DNA)
fluorescence/cell. Computer analysis determined the DNA fragmentation index
(DFI = red fluorescence/total [red and green
fluorescence]). Computer gating defined the X DFI (mean of
DFI ranging from 0 to 1024 channels), the SD DFI (standard deviation of
DFI), the %DFI (percentage of sperm with DNA fragmentation) and the
%HDS (percentage of sperm with high DNA
stainability).
Statistical methods
We used
repeated measures analysis of variance with a compound symmetry correlation
structure to test all two- and three-way interactions between
factors, i.e. donor/patient group, mature/ immature spermatozoa and
normal/abnormal semen parameters. If significant interactions were found,
we assessed one factor within levels of the other.
We compared donors
versus patients examined for infertility and donors with normal semen
parameters versus patients on continuous variables using the Wilcoxon
rank-sum test or Students t-test, as appropriate.
Pairwise comparisons of donors with normal semen parameters,
ROS-negative patients and ROS-positive patients were made on
continuous variables using Dunns or Tukeys multiple
comparison tests. Within donor and patient groups, we compared mature
versus immature spermatozoa on continuous variables using the Wilcoxon
signed-rank test or paired t-test. We assessed the
relationship of apoptosis to ROS and %DFI by computing Spearman
correlation coefficients (r) and corresponding 95%
confidence intervals (CIs) within donor and patient groups. Correlations of
the groups were compared using a z-test for two independent
correlation coefficients (Wilcox,
1987).
ROS-TAC scores were generated through
principal component analysis and standardized so that the mean ROS-TAC
score for the whole ejaculate was 50 and the SD was 10 for the donors with
normal semen parameters (Sharmaet al., 1999). The results are reported as either median
(25th and 75th percentile) or mean (±SD). Two-tailed tests were
performed, and the significance level for each test was 0.05. A Bonferroni
correction was applied to the significance criteria for multiple
comparisons such that P < 0.01 (0.05/5 comparisons)
was considered significant. Analysis was done with SAS 8.2 (SAS Institute
Inc., Cary, NC), and graphics were produced with S-PLUS 6.0
(Insightful Corp.).
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Results |
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Semen parameters (Table I)
Of the 19 donors,
58% (11 of 19) had normal semen parameters. The donors had
significantly higher sperm concentration and percentage motility than the
patients. The 11 donors with normal semen parameters had:
(i) significantly higher sperm concentration, percentage motility and
normal forms as assessed by the WHO guidelines than the patients;
(ii) higher percentage motility and normal forms as assessed by the
WHO guidelines and Krugers strict criteria than the
ROS-negative patients; and (iii) higher sperm concentration and
percentage motility than the ROS-positive patients. The
ROS-negative patients had significantly higher sperm concentration
than the ROS-positive patients.
Apoptosis and necrosis (Table II)
The patients had a significantly higher
percentage of apoptosis in the whole ejaculate than the donors and the
donors with normal semen parameters. The ROS-positive patients had a
higher percentage of apoptosis in the whole ejaculate than the donors with
normal semen parameters and ROS-negative patients. Moreover, the
ROS-negative patients had a higher percentage of apoptosis in the
whole ejaculate than the donors with normal semen parameters. Within every
donor/patient group or subgroup, the percentage of spermatozoa that
underwent apoptosis was higher in the mature spermatozoa than in the
immature spermatozoa.
Patients had a significantly higher percentage of necrosis in the whole ejaculate than the donors as a whole and the donors with normal semen parameters. The ROS-positive and -negative patients had a higher percentage of necrosis than donors with normal semen parameters in the whole ejaculate. Within every donor/patient group or subgroup except for the ROS-positive patients, the immature spermatozoa had a higher percentage of necrosis than mature spermatozoa.
ROS and TAC (Table III)
The patients
had significantly higher levels of ROS in the whole ejaculate and immature
spermatozoa than the donors. As expected, the ROS-positive patients
had higher ROS levels in the whole ejaculate and mature and immature
spermatozoa than the donors with normal semen parameters and the
ROS-negative patients. The immature spermatozoa had higher levels of
ROS than the mature spermatozoa within donors, patients and
ROS-negative patients. Levels of TAC were not significantly different
between any of the groups that were compared.
The donors as a whole and donors with normal semen parameters had significantly higher ROS-TAC scores in the whole ejaculate and immature spermatozoa than the patients. Donors with normal semen parameters and ROS-negative patients had higher ROS-TAC scores in the whole ejaculate and immature spermatozoa than the ROS-positive patients. Within patients and the ROS-positive and -negative patient subgroups, ROS-TAC scores were higher in the mature spermatozoa than in the immature spermatozoa
DNA damage (Table IV)
The
ROS-positive patients had significantly higher %DFI in the
whole ejaculate than the donors with normal semen parameters. There were no
statistically significant differences in the %DFI between the donors
and patients or between the mature and immature spermatozoa within each
subgroup. The ROS-positive patients had a significantly higher mean
and SD DFI in the whole ejaculate than the donors with normal semen
parameters. Within donors, the immature spermatozoa had higher mean and SD
DFI than the mature spermatozoa. There were no significant differences in
%HDS between any of the donor and patient groups or subgroups
compared. Within each donor and patient group or subgroup except for the
ROS-positive patients, the immature spermatozoa had significantly
higher %HDS than the mature
spermatozoa.
Relationship between apoptosis and ROS
Figure 1
illustrates the relationship between apoptosis and ROS within the donor and
patient groups. Apoptosis and ROS were not significantly correlated within
the donors. Apoptosis was significantly correlated with ROS within patients
in the whole ejaculate [r (95%
CI) = 0.53 (0.190.86)] and in the mature
[r (95% CI) = 0.71
(0.391.00)] and immature spermatozoa [r
(95% CI) = 0.75 (0.451.00)]. Patients
had higher absolute value correlation coefficients than donors in the whole
ejaculate, mature and immature spermatozoa.
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Discussion |
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To examine these issues, ROS-positive and
-negative samples from donors with both normal and abnormal semen
parameters were fractionated into mature and immature subsets and then
examined for the extent of apoptosis and DNA damage. The primary function
of density gradient separation is to separate the population of sperm with
greatest fertility potential. In our earlier study (Gil-Guzman et al., 2001), using
a three-layer density gradient (47, 70 and 90%), we identified
the immature and mature spermatozoa in the different subsets separated.
These were identified by staining both for morphological abnormalities and
for the presence of spermatozoa with excessive residual cytoplasm.
We
demonstrated that immature spermatozoa produce significantly higher levels
of ROS and DNA damage (Gil-Guzman et al., 2001;
Ollero et al.,
2001
). In the present study, mature spermatozoa (sperm
fractions from the lower 90% layer of the Percoll gradient) showed
lower generation of ROS and markedly superior sperm motion characteristics.
Forty-one percent of the patients in our study (12 of 29) had
high ROS levels. The higher levels of ROS among the patients examined for
infertility imply that ROS is a significant contributor of male
infertility.
Functionally abnormal spermatozoa have been considered
to be the main source of ROS. However, other cells, particularly
leukocytes, can also generate ROS. Activated leukocytes are capable of
producing 100-fold higher amounts of ROS than non-activated
leukocytes (Plante et al,
1994). Oxidative injury to spermatozoa is a major cause of
sperm dysfunction (Oehninger et
al., 1995
; Sharma
and Agarwal, 1996
; Ollero et al., 2001
; Agarwal and Saleh, 2002
; Alvarez et al., 2002
;
Saleh et al.,
2002a
,b; Agarwal et
al., 2003
).
We did not observe a significant
reduction in the total antioxidant capacity associated with increased
levels of ROS. The pathological levels of ROS detected in the semen of
infertile men are more likely to be caused by increased ROS production than
by reduced antioxidant capacity of seminal plasma (Zini et al.,
1993). Both ROS and TAC markers by themselves are not good discriminators
of oxidative stress. Low ROS-TAC scores in our study indicate high
seminal oxidative stress. This score may serve as an important measure in
identifying men with a clinical diagnosis of infertility who are likely to
initiate a pregnancy over time (Sharma et al., 1999; Pasqualotto et al.,
2000
).
The mechanism of DNA damage in a mature
spermatozoon that is transcriptionally inactive is unclear. It is critical
to understand how DNA damage occurs. Anomalies in the DNA of ejaculated
human spermatozoa can occur in two ways. The first theory is the unique
manner in which sperm chromatin is packaged, while the second attributes
the nuclear DNA damage in mature spermatozoa to apoptosis (Sakkas et al., 2002). At
the nuclear level, histones are replaced by protamines in a process called
protamination. To achieve this purpose, nicks must be created endogenously
to relieve the torsional stress of the DNA double helix. The endogenous
nuclease, topoisomerase II (topo II), might play a role in both creating
and ligating nicks during spermiogenesis (McPherson and Longo, 1992
, 1993
,b; Sakkas et al., 1995
). The presence
of endogenous nicks in ejaculated spermatozoa indicates incomplete
maturation during spermiogenesis. Once protamination is complete, the nicks
completely disappear (Ward and
Coffey, 1991
). Disruption of the critical process of chromatin
packaging may result in persistence of endogenous nicks that would, in
turn, be reflected as DNA damage.
The second theory proposes that the
presence of endogenous nicks in ejaculated human spermatozoa is
characteristic of programmed cell death as seen in apoptosis of somatic
cells (Gorczyca et al.,
1993a,b). Activation of the endogenous endonuclease, which
causes extensive DNA breakage, may therefore represent a ubiquitous
mechanism of cell inactivation (death), or functional elimination of
possibly defective germ cells from the reproductive pool.
The
evidence points to an abortive apoptosis taking place in many males that
exhibit sperm parameters that are below normal. Apoptosis in mature sperm
is initiated during spermatogenesis in which some cells, earmarked for
elimination, may escape the removal mechanism and contribute to poor sperm
quality (Sakkas et al.,
2002; Shen et
al., 2002
). In certain males, abortive apoptosis may fail
in the total clearance of spermatozoa earmarked for elimination by
apoptosis. One of the factors implicated in apoptosis is the cell surface
protein, Fas. Results indicate that subfertile men have increased presence
of Fas-positive spermatozoa (Sakkas et al., 2002
). In these
subfertile men, spermatozoa that have been earmarked to undergo apoptosis
escape this process, suggesting that the correct clearance of spermatozoa
via apoptosis is not occurring.
Apoptotic spermatozoa showed variable
degrees of annexin-V positivity, depending on the stage of the
apoptotic process. This staining pattern depends on the degree of external
expression of the phospholipid PS. As cells progress through the stages of
apoptosis, higher amounts of PS are expressed on the cell surface. In
general, the external expression of PS takes place in an early stage of
apoptosis (Vermes et al.,
1995). Simultaneous staining with PI for necrotic spermatozoa
allows for differentiation.
Annexin V staining is a physiological
marker of early apoptosis; however, it is also associated with
bicarbonate-mediated phospholipid scrambling during capacitation
(Gadella and Harrison,
2002; de Vries et
al., 2003
). There fore, it is important to complement
annexin-V staining with other markers of DNA damage such as TUNEL
(strand breaks) or DNA fragmentation by SCSA, both of which are highly
correlated (Sailer et al.,
1995
). We therefore also examined the extent of DNA damage in
these specimens utilizing SCSA as a measure of DNA damage. In our study,
apoptosis occurred in spermatozoa from donors with normal semen parameters
and also in spermatozoa from patients examined for infertility at increased
levels, reflecting the significant role of this process in male
infertility.
A number of studies using TUNEL, Fas binding on mature
spermatozoa and annexin-V staining have proposed that the presence
of spermatozoa with damaged DNA is indicative of apoptosis (Manicardi et al., 1995,
1998
; Sun et al., 1997
;
Lopes et al.,
1998
; Sakkas et
al., 1999
; Barroso et al., 2000
; Irvine et al., 2000
).
Mature spermatozoa had lower levels of DNA damage than immature spermatozoa
in donors with normal semen parameters. However, in patients examined for
infertility, mature spermatozoa had increased levels of DNA damage.
Variable amounts of DNA fragmentation are seen in the high and low motility
fractions of ejaculates from infertile men and normozoospermic donors
(Sun et al.,
1997
; Aitken et
al., 1998
; Lopes et al., 1998
; Barroso et al., 2000
;
Donnelly et al.,
2000
; Oosterhuis et
al., 2000
). The differences in the proportion of sperm
with DNA fragmentation among all these studies may be due to the sperm
samples analysed (donors versus different patient populations), the various
sperm separation methods, and the different methods used to detect DNA
fragmentation.
This is important as mature spermatozoa with DNA damage may exhibit lower functional potential and this may explain the patients subfertility status. Furthermore, it raises the concern of using such spermatozoa during ICSI. We found that the level of DNA damage was higher in patients examined for infertility in our study.
ROS and their role in male infertility have been researched extensively. Many studies have shown the adverse effects of ROS on the different cellular compartments of spermatozoa including DNA. ROS can result in single- and double-strand DNA breaks. Impaired chromatin packaging, apoptosis and ROS alone can induce DNA damage in human spermatozoa. Thus, a causeeffect relationship exists between apoptosis and DNA damage. This explains the positive correlation between apoptosis and DNA damage observed in our study.
However, comparing levels of apoptosis with
those of DNA damage in the same sample has indicated that apoptosis might
be responsible only for a fraction of DNA damage in human spermatozoa.
Additionally, we have observed a strong correlation between ROS and the
level of apoptosis. This may reflect a causal relationship in the way that
ROS can induce apoptosis. Our results are supported by a recent publication
by Sakkas et al.
(2002) in which they reported that TUNEL positivity and
apoptotic markers do not always exist in unison in spermatozoa. In this
study, semen samples that had low sperm concentration and poor morphology
were more likely to show high levels of TUNEL positivity and Fas and p53
expression. However, no strict correlation was evident between Fas and p53
expression and the population of sperm that were positive for TUNEL. The
authors proposed that DNA damage is not directly linked to an apoptotic
process occurring in spermatozoa, and may arise due to problems in the
nuclear remodelling process during the later stages of spermatogenesis
(Sakkas et al.,
2002
).
In our study, we have demonstrated a strong correlation between apoptosis and sperm DNA damage in the whole ejaculate of our patient group. However, the total amount of the DNA damage observed in our study cannot be explained by apoptosis alone. Furthermore, not all apoptotic spermatozoa observed in our study possessed DNA damage. A proportion of them should be in an early stage of apoptosis. Thus, apoptosis can explain only a fraction of such DNA damage.
The role
for caspases and apoptosis in ejaculated sperm is still in question.
Caspase, c-jun, p53 and p21 are present in a restricted site for
apoptosis (cytoplasmic droplets) in spermatids and immature spermatozoa
(Weil et al.,
1998; Blanco-Rodriguez and Martinez-Garcia,
1999
). Inactive and active form of caspase markers have been
detected in human sperm cells (Weng et al., 2002
; de Vries et al., 2003
) in
both low and high motility fractions of donors and patients. A significant
positive correlation has been shown between in situ-active
caspase 3 in the sperm midpiece and DNA fragmentation in the low motility
fractions of patients. This suggests that caspase-dependent apoptotic
mechanisms could originate in the cytoplasmic droplet or within
mitochondria, and function in the nucleus (Weng et al., 2002
).
Mature
sperm do not have efficient operative mechanisms for protein synthesis.
Both active and inactive forms of caspases (caspase 3) are absent in mature
sperm cells (de Vries et
al., 2003). They do not show
bicarbonate/PKA-dependent signs of apoptosis such as fractionation of
DNA or mitochondrial inner membrane depolarization, but do show rapid
aminophospholipid exposure (de
Vries et al., 2003
). There may be a temporal
disassociation between caspase activation and the expression of cellular
changes suggestive of apoptosis in mature spermatozoa.
On the other
hand, Paasch et al.
(2003) recently reported that active caspases were present in
subpopulations of mature sperm and to a greater extent in sperm from
infertile patients. However, caspases are not completely removed during
undisturbed spermatogenesis and may therefore contribute to inhibition of
normal sperm function. This finding offers new insight into the current
concepts in remodelling defects during spermatogenesis.
Sperm cells
with immature appearance and/or cytoplasmic droplets fail to expose PS and
also show no phosphotyrosine labelling (de Vries et al., 2003).
Alternatively, triggering of PS externalization and DNA fragmentation could
be due to activation of other caspases or cellular pathways. It also leaves
open the possibility that sperm apoptosis may be caspase independent, at
least to some extent. Our data also support this observation since we did
not find a significant correlation of apoptosis with DNA damage, suggesting
a nuclear remodelling phenomenon rather than apoptosis as a cause of
ROS-induced DNA damage.
From all of these observations, we conclude that ROS generation contributes to DNA damage in spermatozoa. Mature spermatozoa had lower levels of DNA damage compared with immature spermatozoa in our group of normal men who serve as volunteers for our research studies. In patients examined for infertility, however, there may be an increased number of spermatozoa with DNA damage, particularly in the mature fraction, explaining their subfertility. The contributing role of each one of these mechanisms will vary among individuals and within a given individual from time to time depending on the circumstances that affect spermatogenesis. The patients in our study consisted of men presenting with a history of infertility at our infertility clinic. These men could not be characterized as being infertile (or not), due to a lack of information on either male or female factors, and therefore may have included men with apparently normal semen.
The observation that ejaculated human spermatozoa possess DNA damage raises numerous problems. How these spermatozoa arise in the ejaculate of some men and what consequences they have if they succeed in their genetic mission are unclear. Further investigations are required to assess the potential capacity of ejaculated human spermatozoa to undergo apoptotic cell death. It is unclear why the apoptotic process is not fully completed in these sperm.
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Submitted on May 29, 2003; resubmitted on July 24, 2003; accepted on September 29, 2003.