1 Department of Community, Occupational and Family Medicine, Faculty of Medicine, National University of Singapore, Republic of Singapore, 2 School of Public Health, Sun Yat-sen University, China and 3 Department of Obstetrics and Gynaecology, Singapore General Hospital, Republic of Singapore
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Abstract |
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Key words: Annexin-V/apoptosis/infertile/sperm/TUNEL
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Introduction |
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In contrast, apoptosis in ejaculated sperm is less well studied. Gorczyca et al. were among the first to suggest that DNA breaks in abnormal sperm were analogous to apoptosis in somatic cells (Gorczyca et al., 1993). Subsequently, morphological evidence was provided for the presence of characteristic apoptotic ultrastructural alterations in ejaculated sperm cells (Baccetti et al., 1996
). Some recent studies attempted to link apoptotic cell death in sperm with conventional seminal parameters (Sun et al., 1997
; Gandini et al., 2000
; Irvine et al., 2000
). However, the influence of apoptosis on sperm function and quality is largely elusive. Furthermore, many of the reports studied DNA strand breaks as the main evidence for apoptosis in sperm (Barroso et al., 2000
; Gandini et al., 2000
; Muratori et al., 2000
; Oosterhuis et al., 2000
). Although the detection of phosphatidylserine (PS) exposure, a well established early apoptosis marker, has been applied in the study of sperm apoptosis (Barroso et al., 2000
; Oosterhuis et al., 2000
), it is not known whether such changes are associated with apoptotic DNA strand breaks in mature sperm. In fact, one recent report failed to link DNA fragmentation in ejaculated sperm with other apoptotic phenomenon observed at submicroscopic level (Muratori et al., 2000
). Therefore, the objectives of the present study are (i) to examine the apoptotic alterations in ejaculated sperm in a group of subfertile patients, by using both Annexin-V staining for the detection of membrane PS externalization and Tdt-mediated dUTP nick end labelling (TUNEL) for the measurement of DNA fragmentation, and (ii) to further evaluate the functional impact of sperm apoptosis on sperm quality, especially its correlation with sperm defects.
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Materials and methods |
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Detection of membrane PS exposure
PS is normally restricted to the inner leaflet of the plasma membrane bilayer. Apoptosis causes membrane phospholipid asymmetry and translocation of PS onto the outer leaflet of the membrane, and the detection of PS exposure has been well established as an early apoptotic marker (Vermes et al., 1995). In the present study, detection of PS externalization in sperm was performed using an Annexin-V-fluos staining kit (Roche, Indianapolis, IN, USA), following the manufacturer's protocol with modifications. Generally, semen samples containing 1x106 sperm were first washed twice (200 g for 10 min, 4°C) with EGTA/Hepes A buffer (containing 1 mmol/l EGTA, 137 mmol/l NaCl, 2.68 mmol/l KCL, 10 mmol/l Hepes, 1.7 mmol/l MgCl2, 25 mmol/l glucose; pH7.4), followed by a single wash with Hepes A buffer. The washed sperm were resuspended in the Annexin-V labelling solution [containing recombinant Annexin-V protein, Ca2+ and propidium iodide (PI) in Hepes buffer]. After incubation at room temperature for 10 min, sperm were analysed immediately by flow cytometry (Coulter Epics Elite ESP, Miami, FL, USA) using 488 nm excitation and a 515 nm bandpass filter for FITC detection and a 610 nm bandpass filter for PI detection. At least 10 000 cells were examined for each sample and the test was run in duplicate for each subject. Obtained data were analysed using WinMDI 2.7 software (Scripps Institute, La Jolla, CA, USA) for calculating the percentage of normal (both Annexin-V- and PI-negative), early apoptotic (Annexin-V-positive and PI-negative), and late apoptotic or secondarily necrotic cells (both Annexin-V- and PI-positive). A negative control was also included for each batch without the presence of Annexin-V in the labelling solution. In addition, the stained cells were also examined under a confocal microscope (Zeiss LSM 410, equipped with a Plan-APOCHROMAT 63x/1,40 oil DIC objective lens) for the morphological changes.
TUNEL assay
TUNEL assay has been well established for the detection of DNA cleavage during apoptosis (Heatwole, 1999). Recently this technique has also been widely used to determine DNA damage in human sperm (Sailer et al., 1995
; Aravindan et al., 1997
; Sun et al., 1997
). In the present study, the TUNEL assay in human sperm was conducted in our laboratory using the technique described earlier (Shen and Ong, 2000
), using an in-situ cell death detection kit (Roche, Indianapolis, IN, USA). About 2x106 sperm were first washed with Hepes A buffer as described above, and then fixed in 2% paraformaldehyde for 30 min at room temperature. The fixed cells were resuspended in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 10 min on ice. After washing with Hepes A buffer, the cells were gently resuspended in the Tdt reaction solution containing Tdt enzyme and FITC-labelled 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 37°C in the dark, the sample was analysed using flow cytometry with an air-cooled argon 488 nm laser and a 550 nm dichroic mirror. At least 10 000 cells in each sample were collected and the percentage of TUNEL-positive cells were calculated from the histogram using the WinMDI 2.7 software.
Statistical analysis
Pearson's correlation coefficients were calculated to determine the correlation between the apoptotic indices and routine semen parameters after logarithmic transformation, using SPSS version 10.0 software for Windows (SPSS Inc., Chicago, IL, USA).
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Results |
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Discussion |
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In this study, apoptotic alterations in sperm were further examined using the TUNEL assay. On average about 15% of sperm were found to be TUNEL-positive, a value similar to a number of other reports conducted on infertile patients (Lopes et al., 1998b; Oosterhuis et al., 2000
). Moreover, it is interesting to note that the percentage of TUNEL (+) cells only significantly correlated with double positive cells [Annexin-V (+)/PI (+)], but not with only Annexin-V (+) cells (Figure 3
). This can be explained by the common understanding that DNA fragmentation usually occurs at the late stage of apoptosis caused by endonuclease activation (Arends et al., 1990
). Such correlations also suggest that the double positive cells [Annexin-V (+)/PI (+)] are more likely to be late apoptotic cells, rather than necrotic cells. Results from this study thus indicate that both Annexin-V staining for the identification of PS exposure and the TUNEL assay for the measurement of DNA fragmentation are valid tools for the assay of apoptosis in sperm.
The study of various forms of DNA damage in human sperm has a much longer history than the study of sperm apoptosis. Various techniques have been developed for examining such damage, including TUNEL, in-situ nick translation, and single cell gel electrophoresis (comet assay) (Bianchi et al., 1993; Gorczyca et al., 1993
; McKelvey-Martin et al., 1997
; Aravindan et al, 1997
; Singh and Stephens, 1998; Shen and Ong, 2000
). Although it is acknowledged that the extent of sperm DNA damage is closely related to sperm function and male infertility (Aitken, 1999
; Sakkas et al., 1999b
), the origin of such DNA damage in mature sperm is still largely controversial. Three hypotheses have been postulated to explain the source of DNA damage in sperm. First, it is believed that sperm DNA damage is caused by improper packaging and ligation during sperm maturation (Sailer et al., 1995
). Second, oxidative stress (elevated level reactive oxygen species or diminished antioxidant defence mechanism) causes DNA damage and the increased level of specific form of oxidative DNA damage such as 8-hydroxydeoxyguanosine in sperm DNA supports such a theory (Lopes et al., 1998a
; Aitken, 1999
; Shen and Ong, 2000
). Third, observed sperm DNA fragmentation is caused by apoptosis (Sakkas et al., 1999b
). In the present study, the close correlation between TUNEL-positive cells and Annexin-V stained sperm suggests that apoptosis is an underlying mechanism for the observed DNA damage in this group of subfertile subjects, although contributions from the other two pathways may co-exist.
Clinically, conventional seminal parameters are not always sufficient in the assessment of sperm function and male fertility. Therefore, attempts have been made to link apoptotic alterations in sperm with some conventional sperm parameters. For instance, sperm apoptosis was found to be inversely correlated with sperm motility (Barroso et al., 2000; Oosterhuis et al., 2000
). Similar inverse correlations between apoptotic alterations in sperm and sperm motility as well as vitality were also observed in the present study (Table II
). One the other hand, substantial efforts have been undertaken to link the morphological features of sperm with apoptotic alterations (Baccetti et al., 1996
; Gandini et al., 2000
; Muratori et al., 2000
). For example, Baccetti et al. (1996) evaluated for the first time the ultra structural changes in apoptotic sperm cells. Gandini et al.. (2000) also examined the morphological aspect of the apoptotic sperm together with the TUNEL assay. In the present study, the apoptotic alterations in sperm were positively associated with various forms of abnormal sperm morphology, including the sperm head, mid-piece and tail defects (Figure 4
). Therefore, results from this study further demonstrate the adverse functional impact of sperm apoptosis on sperm quality. Another important finding of the present investigation is that only late apoptosis determined by Annexin-V staining or TUNEL (+) cells were correlated to seminal parameters, and no close correlations were found with early apoptosis. Such results indicate that the sperm function will only be affected at the late stage of apoptosis when both DNA fragmentation and cell membrane damage become evident.
In most other cell types, the apoptotic signalling pathways have been well established. For instance, mitochondria occupy a central position as many apoptosis stimuli converge on mitochondria. The release of pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF) from mitochondria will then initiate the downstream caspases such as caspase 9 or 3 to execute the apoptotic events (Green and Reed, 1998; Desagher and Martinou, 2000
). However, such apoptotic mechanistic pathways have not been observed in mature sperm. It is thus believed that mature sperm lack the ability to undergo `real' apoptosis.
In the present study, the sperm samples from this group of patients had two distinctive features (Table I): (i) most of them were within the normal range of sperm concentration or total of sperm number, and (ii) the percentage of sperm defects was unusually high. To our surprise, the percentage of apoptosis was significantly correlated with both sperm concentration and total sperm count, which is found to be contradictory with two recent reports (Barroso et al., 2000
; Oosterhuis et al., 2000
). One possible explanation for such positive correlations is the abortive apoptosis theory proposed by Sakkas et al. (1999a,b), which is supported by substantial experimental evidence. First, it is known that mature sperm lack the apoptosis machinery such as caspases (Weil et al., 1998
). Therefore, the observed apoptotic alterations including PS exposure and DNA fragmentation are likely to have originated from the apoptotic process which is triggered during spermatogenesis. Second, Fas and Fas ligand are very important regulatory mechanisms for the control of germ cell apoptosis (Lee et al., 1997
; Print and Loveland, 2000
; Kierszenbaum, 2001
). It has been found that the number of sperm with Fas expression was low in subjects with normal sperm parameters but high in men with abnormal sperm parameters (Sakkas et al., 1999a
). The high percentage of apoptotic cells in subfertile patients may imply that more sperm that have been earmarked to undergo apoptosis escape this process owing to an abortive apoptotic mechanism. In the present study, more than 40% of the sperm on average were labelled with Annexin-V (Figure 3
), and these are the cells that should be eliminated if the apoptotic machinery functions properly during the spermatogenesis stage. The positive correlations between the apoptotic indices and various forms of sperm defects including head, mid-piece and tail found in the present study (Figure 4
) also tend to support the above explanation. Therefore, although the presence of defective apoptotic cells does not affect the sperm concentration and sperm counts, they adversely affect sperm quality and functions, and may eventually contribute to infertility.
In summary, the use of both Annexin-V staining and the TUNEL assay data from the present study provide clear evidence that the apoptotic alterations in a group of subfertile subjects are closely correlated to sperm quality such as motility, vitality and sperm defects. The positive correlation between sperm concentration or total sperm counts with apoptotic indices may be explained, to a certain extent, by the abortive apoptosis theory. Those cells destined to undergo apoptosis may escape the clearance mechanism during spermatogenesis and their presence in the ejaculate thus contributes to poor sperm quality.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 10, 2001; resubmitted on August 20, 2001; accepted on January 8, 2002.