Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality

Han-Ming Shen1, Jun Dai2, Sin-Eng Chia1, Alvin Lim3 and Choon-Nam Ong1,,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of the present study was to define the effect of apoptosis on sperm quality and function. METHODS: The apoptotic features in sperm were assessed in 60 subfertile subjects, using Annexin-V staining for phosphatidylserine (PS) externalization and Tdt-mediated dUTP nick end labelling (TUNEL) assay for DNA fragmentation. RESULTS: On average, about 45% of the sperm were found to be apoptotic based on the results from Annexin-V staining, including both early (Annexin-V-positive, PI-negative) and late apoptosis (Annexin-V-positive, PI-positive). TUNEL-positive cells (median value 15%) significantly correlated to late apoptosis but not early apoptosis, indicating that DNA fragmentation only occurs at the later stage of sperm apoptosis. TUNEL-positive and late apoptotic cells (Annexin-V-positive, PI-positive) were found to be inversely correlated to sperm motility and vitality, and positively to abnormal sperm morphology. On the other hand, it is surprising to note that the apoptotic alterations in sperm positively correlated to sperm concentration or total sperm counts. CONCLUSIONS: Overall results from this study support the abortive apoptosis theory; apoptosis in mature sperm is initiated during spermatogenesis, after which some cells earmarked for elimination via apoptosis may escape the removal mechanism and contribute to poor sperm quality.

Key words: Annexin-V/apoptosis/infertile/sperm/TUNEL


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Apoptosis, or programmed cell death, is a distinctive form of cell death characterized by a series of morphological and biochemical changes that result in efficient elimination of cells from tissues without eliciting an inflammatory response (Wyllie, 1980Go). It is well known that apoptosis plays a critical role in many physiological and pathological processes such as development, tissue homeostatasis, and diseases (Bellamy et al., 1995Go). Recently, the role of apoptosis in spermatogenesis has attracted substantial research interest. Spermatogenesis refers to a dynamic process encompassing the mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, the morphological differentiation and maturation of spermatids, and finally the formation of sperm (Print and Loveland, 2000Go). It has been estimated that in human testes, one spermatogonia gives rise only to about 100 spermatids, far below the theoretical value of 4096 (Woolveridge and Morris, 2000Go), indicating the involvement of physiological cell death in this process. Currently it is commonly understood that germinal cell apoptosis is an underlying mechanism for normal spermatogenesis (Print and Loveland, 2000Go; Kierszenbaum, 2001Go). In human testicular biopsies, a certain degree of spontaneous apoptosis was observed (Brinkworth et al., 1997Go). On the other hand, an altered apoptotic process has been found to be closely associated with male infertility, based on the following observations: (i) men with azoospermia or severe oligozoospermia have an increased frequency of apoptotic germ cells in their testicular tissues in comparison with those with normal spermatogenesis (Lin et al., 1997aGo,bGo; Sinha Hikim et al., 1998Go); and (ii) some reproductive toxicants such as cadmium are able to induce germ cell apoptosis (Richburg, 2000Go). Moreover, experiments with transgenic animals have also demonstrated that those animals with apoptosis factors such as Bax deficiency showed increased level of apoptosis in germ cells and impaired fertility (Knudson et al., 1995Go; Rodriguez et al., 1997Go).

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., 1993Go). Subsequently, morphological evidence was provided for the presence of characteristic apoptotic ultrastructural alterations in ejaculated sperm cells (Baccetti et al., 1996Go). Some recent studies attempted to link apoptotic cell death in sperm with conventional seminal parameters (Sun et al., 1997Go; Gandini et al., 2000Go; Irvine et al., 2000Go). 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., 2000Go; Gandini et al., 2000Go; Muratori et al., 2000Go; Oosterhuis et al., 2000Go). 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., 2000Go; Oosterhuis et al., 2000Go), 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., 2000Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Subjects and routine semen analysis
Patients who visited the Fertility Clinic of the Singapore General Hospital during the period of September to October 2000 were recruited for the present study. The study was approved by the ethics committee for research involving human subjects at the Faculty of Medicine, National University of Singapore. Semen samples were collected by masturbation after 3–5 days of sexual abstinence. They were allowed to liquefy for 30 min at room temperature. The liquefied samples were then divided into two parts, one for routine parameter analysis and another for assessment of apoptosis. The routine seminal parameters were evaluated according to the World Health Organization (WHO) Criteria (World Health Organization, 1999Go). The variables taken into consideration were: volume, sperm concentration, total sperm number, motility, vitality, normal morphology, and sperm defects. Sperm morphology was assessed according to strict criteria (Menkveld et al., 1990Go; World Health Organization, 1999Go). Briefly, semen smears were made on microscope slides and then treated with the Papanicolaou staining protocol. The slides were read under brightfield microscopy with x1000 magnification under oil (Nikon SE, Tokyo, Japan). Sperm were considered morphologically normal only if the head, mid-piece and tail were all normal. Head defects were mainly amorphous, tapering (elongated) and small (<4 µm length), while mid-piece and tail defects were mainly seen as enlargement and tail coiling respectively.

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., 1995Go). 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, 1999Go). Recently this technique has also been widely used to determine DNA damage in human sperm (Sailer et al., 1995Go; Aravindan et al., 1997Go; Sun et al., 1997Go). In the present study, the TUNEL assay in human sperm was conducted in our laboratory using the technique described earlier (Shen and Ong, 2000Go), 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 60 subjects visiting the Fertility Clinic during the study period were recruited. Their ages ranged from 25 to 45 years and most of them were non-smokers (data not shown). According to reference values of WHO (World Health Organization, 1999Go), the majority of the subjects were within the normal ranges of semen volume, concentration, and total sperm number (73.3%, 86.7% and 91.7% within normal ranges respectively) (Table IGo). The median value of sperm concentration found in this study is also similar to the reported value of a local confirmed fertile population (Chia et al., 1998Go). The present study adopted more strict criteria (World Health Organization, 1999Go) for the assessment of sperm morphology, which may explain the obviously low percentage of sperm with normal morphology in this group of subjects (ranging from 1–16%). The median value of normal morphology among the 60 subjects was only 7%, far below the WHO suggested value (15%, World Health Organization, 1999Go). Therefore, poor sperm morphology appears to be one of the main characteristics of our study population.


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Table I. Characteristics of seminal parameters
 
In the present study, two assays were applied to examine the apoptotic alterations in ejaculated sperm: Annexin-V staining for determination of PS exposure and TUNEL for identification of DNA fragmentation. Figure 1AGoshows the typical dotplots of Annexin-V and PI staining in which three distinctive cell populations can be clearly seen: normal cells with both Annexin-V and PI staining negative, early apoptotic cells with Annexin-V-positive but PI-negative, late apoptotic or secondarily necrotic cells with both Annexin-V and PI-positive (Figure 1AGo, I–III). Cells were also examined under a laser confocal microscope and the results are presented in Figure 1BGo. Sperm defects such as enlarged mid-piece were evident (Figure 1BGo, arrows). The translocation of PS was demonstrated by the staining of Annexin-V conjugated FITC green fluorescence around the head, mid-piece, and certain parts of the tail, while the staining of the sperm head by PI red fluorescence indicates the damage of membrane integrity (Figure 1BGoII).



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Figure 1. Detection of apoptosis in spermatozoa using Annexin-V staining. (A) Data from flow cytometry: negative control in which virtually no cells stained positive (I), samples with low percentage of apoptosis (II) and samples with high percentage of apoptosis (III). Three cell populations were distinctive: normal cells (both Annexin-V- and PI-negative), early apoptotic cells [Annexin-V (+)/PI (-)], and late apoptotic or secondarily necrotic cells [Annexin-V (+)/PI (+)]. (B) Transmissional (I) and confocal images (II) showing both Annexin-V staining (green colour) and PI staining (red). Arrows point to obvious morphological alterations such as mid-piece defects.

 
Figure 2Godemonstrates the typical histograms of the TUNEL assay analysed by flow cytometry. Compared with the negative control (Figure 2AGo), a significant increase of TUNEL-positive cells was observed in the samples (Figure 2BGo). When cells were treated with DNase I as positive control, about 80% of cells were constantly observed to be TUNEL-positive (data not shown), which is consistent with our earlier report (Shen and Ong, 2000Go).



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Figure 2. Detection of apoptosis in spermatozoa using Tdt-mediated dUTP nick end labelling (TUNEL) assay. Histogram data from flow cytometry of a negative control (A) and one sample with 40% cells labelled with high FITC fluorescence as TUNEL-positive cells (B).

 
When the two methods were compared, Annexin-V staining appeared to be more sensitive than the TUNEL assay. As shown in Figure 3AGo, more than 40% of the cells were labelled with Annexin-V. The median values for Annexin-V (+)/PI (-) (early apoptosis) and Annexin-V (+)/PI (+) (late apoptosis) were 24.7 and 19.7% respectively and both were higher than that obtained by the TUNEL assay (15.0%). It was also interesting to note that the percentage of late apoptosis significantly correlated with early apoptosis (r = 0.451, P < 0.01), and with TUNEL-positive cells (r = 0.546, P < 0.01). In contrast, no correlation was observed between early apoptosis and TUNEL-positive cells (r = 0.158).



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Figure 3. Comparison among results obtained from bothAnnexin-V staining and TUNEL assay. (A) Median values ofboth Annexin-V (+)/PI (-) (early apoptosis), Annexin-V (+)/PI (+) (late apoptosis), and TUNEL (+) cells. Number on the top of each bar is the range of each parameter. (B) Correlation between early apoptosis and late apoptosis. C. Correlation between late apoptosis and TUNEL (+) cells. In both (B) and (C), two-tailed Pearson's correlation coefficients were calculated after logarithmic transformation.

 
In order to assess the possible impact of sperm apoptosis on sperm quality, we further analysed the correlations between the apoptotic indices and various seminal parameters, and the results are summarized in Table IIGo. It is interesting to note that both Annexin-V (+)/PI (+) (late apoptosis) and TUNEL (+) cells were positively correlated to concentration, total sperm number and sperm defects. Moreover they were also inversely correlated to sperm vitality and motility. In contrast, no correlation was observed between the early apoptotic index [Annexin-V (+)/PI (-)] and all these seminal parameters, suggesting that sperm quality was only affected at the late stage of apoptosis. As shown in Table IGo, one of the most remarkable changes of seminal parameters in the present study is the abnormal sperm morphology (median value for normal morphology was only 7%). Thus we further examined the association of apoptosis with different forms of sperm defects and the results are shown in Figure 4Go. Positive correlations were found for both Annexin-V (+)/PI (+) cells and TUNEL (+) cells with head, mid-piece and tail defects, indicating a close association of sperm apoptosis with sperm defects.


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Table II. Correlations of sperm apoptosis with seminal parameters
 


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Figure 4. Correlations between sperm apoptosis and sperm defects. Positive correlations were found between late apoptotic cell death and the percentage of sperm with head defects (A), mid-piece defects (B) and tail defects (C). Similar correlations were also observed between TUNEL (+) cells and various forms of sperm defects (DF). Two-tailed Pearson's correlation coefficients were calculated after logarithmic transformation.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The biochemical events of apoptosis have been well defined. One of the earliest alterations is the disturbance of cell membrane symmetry and the translocation of PS to the outer leaflet of the plasma membrane (Vermes et al., 1995Go). At present, the Annexin V-staining assay, in conjunction with a vital dye (PI), has been developed as a sensitive method for the detection of progressive stage of apoptosis: early apoptosis (Annexin-V-positive and PI-negative) and late apoptosis/ secondary necrosis (both Annexin-V- and PI-positive) (Boersma et al., 1996; Zhang et al., 1997). In the present study, two stages of apoptosis were identified: early apoptosis [Annexin-V (+)/PI (-)] and late apoptosis or secondary necrotic cells [Annexin-V (+)/PI (+)], with a median value of about 20% for each category. Such findings were generally consistent with data from early reports using a similar approach (Barroso et al., 2000Go; Oosterhuis et al., 2000Go). In addition to mature sperm, significant PS externalization has been observed in various stages of apoptotic germ cells (Shiratsuchi et al., 1997Go; Tesarik et al., 1998Go). It is not known whether such membrane alteration originated from the process of spermatogenesis, rather than apoptosis. Therefore, the functional implication of PS translocation in sperm biology remains to be further investigated.

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., 1998bGo; Oosterhuis et al., 2000Go). 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 3Go). 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., 1990Go). 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., 1993Go; Gorczyca et al., 1993Go; McKelvey-Martin et al., 1997Go; Aravindan et al, 1997Go; Singh and Stephens, 1998; Shen and Ong, 2000Go). Although it is acknowledged that the extent of sperm DNA damage is closely related to sperm function and male infertility (Aitken, 1999Go; Sakkas et al., 1999bGo), 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., 1995Go). 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., 1998aGo; Aitken, 1999Go; Shen and Ong, 2000Go). Third, observed sperm DNA fragmentation is caused by apoptosis (Sakkas et al., 1999bGo). 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., 2000Go; Oosterhuis et al., 2000Go). Similar inverse correlations between apoptotic alterations in sperm and sperm motility as well as vitality were also observed in the present study (Table IIGo). One the other hand, substantial efforts have been undertaken to link the morphological features of sperm with apoptotic alterations (Baccetti et al., 1996Go; Gandini et al., 2000Go; Muratori et al., 2000Go). 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 4Go). 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, 1998Go; Desagher and Martinou, 2000Go). 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 IGo): (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., 2000Go; Oosterhuis et al., 2000Go). 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., 1998Go). 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., 1997Go; Print and Loveland, 2000Go; Kierszenbaum, 2001Go). 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., 1999aGo). 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 3Go), 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 4Go) 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors would like to thank Ms Lee Mee Ho and Ms Hum Siew Chen at the Department of Obstetrics and Gynaecology, Singapore General Hospital, and Mr Hy Ong and Mr Ny Khoo at the Department of COFM, NUS for their excellent technical support.


    Notes
 
4 To whom correspondence should be addressed at: Department of Community, Occupational and Family Medicine, Faculty of Medicine, MD3, National University of Singapore, 16 Medical Drive, Singapore 117597, Republic of Singapore. E-mail: cofongcn{at}nus.edu.sg Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Aitken, R.J. (1999) The human spermatozoon – a cell in crisis? J. Reprod. Fertil., 115, 1–7.[Abstract]

Aravindan, G.R., Bjordahl, J., Jost, L.K. and Evenson, D.P. (1997) Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp. Cell Res., 236, 231–237.[ISI][Medline]

Arends, M.J., Morris, R.G. and Wyllie, A.H. (1990) Apoptosis. The role of the endonuclease. Am. J. Pathol., 136, 593–608.[Abstract]

Baccetti, B., Collodel, G. and Piomboni, P. (1996) Apoptosis in human ejaculated sperm cells (Notulae seminologicae 9). J. Submicrosc. Cytol. Pathol., 28, 587–596.[ISI][Medline]

Barroso, G., Morshedi, M. and Oehninger, S. (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum. Reprod., 15, 1338–1344.[Abstract/Free Full Text]

Bellamy, C.O., Malcomson, R.D., Harrison, D.J. and Wyllie, A.H. (1995) Cell death in health and disease: the biology and regulation of apoptosis. Semin. Cancer Biol., 6, 3–16.[ISI][Medline]

Bianchi, P.G., Manicardi, G.C., Bizzaro, D., Bianchi, U. and Sakkas, D. (1993) Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biol. Reprod., 49, 1083–1088.[Abstract]

Brinkworth, M.H., Weinbauer, G.F., Bergmann, M. and Nieschlag, E. (1997) Apoptosis as a mechanism of germ cell loss in elderly men. Int. J. Androl., 20, 222–228.[ISI][Medline]

Chia, S.E., Tay, S.K. and Lim, S.T. (1998) What constitutes a normal seminal analysis? >Semen parameters of 243 fertile men. Hum. Reprod., 13, 3394–3398.[Abstract]

Desagher, S. and Martinou, J.C. (2000) Mitochondria as the central control point of apoptosis. Trends Cell Biol., 10, 369–377.[ISI][Medline]

Gandini, L., Lombardo, F., Paoli, D., Caponecchia, L., Familiari, G., Verlengia, C., Dondero, F. and Lenzi A. (2000) Study of apoptotic DNA fragmentation in human spermatozoa. Hum. Reprod., 15, 830–839.[Abstract/Free Full Text]

Gorczyca, W., Traganos, F., Jesionowska, H. and Darzynkiewicz, Z. (1993) Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp. Cell Res., 207, 202–205.[ISI][Medline]

Green, D.R. and Reed, J.C. (1998) Mitochondria and apoptosis. Science, 281, 1309–1312.[Abstract/Free Full Text]

Heatwole, V.M. (1999) TUNEL assay for apoptotic cells. Methods Mol. Biol., 115, 141–148.[Medline]

Irvine, D.S., Twigg, J.P., Gordon, E.L., Fulton, N., Milne, P.A. and Aitken, R.J. (2000) DNA integrity in human spermatozoa: relationships with semen quality. J. Androl., 21, 33–44.[Abstract/Free Full Text]

Kierszenbaum, A.L. (2001) Apoptosis during spermatogenesis: the thrill of being alive. Mol. Reprod. Dev., 58, 1–3.[ISI][Medline]

Knudson, C.M., Tung, K.S., Tourtellotte, W.G., Brown, G.A. and Korsmeyer, S.J. (1995) Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science, 270, 96–99.[Abstract]

Lee, J., Richburg, J.H., Younkin, S.C. and Boekelheide, K. (1997) The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology, 138, 2081–2088.[Abstract/Free Full Text]

Lin, W.W., Lamb, D.J., Wheeler, T.M., Abrams, J., Lipshultz, L.I. and Kim, E.D. (1997a) Apoptotic frequency is increased in spermatogenic maturation arrest and hypospermatogenic states. J. Urol., 158, 1791–1793.[ISI][Medline]

Lin, W.W., Lamb, D.J., Wheeler, T.M., Lipshultz, L.I. and Kim, E.D. (1997b) In situ end-labeling of human testicular tissue demonstrates increased apoptosis in conditions of abnormal spermatogenesis.Fertil. Steril., 68, 1065–1069.[ISI][Medline]

Lopes, S., Jurisicova, A., Sun, J.G. and Casper, R.F. (1998a) Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod., 13, 896–900.[Abstract]

Lopes, S., Sun, J.G., Jurisicova, A., Meriano, J. and Casper, R.F. (1998b) Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil. Steril., 69, 528–532.[ISI][Medline]

McKelvey-Martin, V.J., Melia, N., Walsh, I.K., Johnston, S.R., Hughes, C.M., Lewis, S.E.M. and Thompson, W. (1997) Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1) human bladder washings and transitional cell carcinoma of the bladder; and (2) human sperm and male infertility. Mut. Res., 375, 93–104.[ISI][Medline]

Menkveld, R., Stander, F.S.H, Kotze, T.J.V.W., Kruger, T.F. and van Zyl, J.A. (1990) The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum. Reprod., 5, 586–592.[Abstract]

Muratori, M., Piomboni, P., Baldi, E., Filimberti, E., Pecchioli, P., Moretti, E., Gambera, L., Baccetti, B., Biagiotti, R., Forti, G. and Maggi, M. (2000) Functional and ultrastructural features of DNA-fragmented human sperm. J. Androl., 21, 903–912.[Abstract/Free Full Text]

Oosterhuis, G.J., Mulder, A.B., Kalsbeek-Batenburg, E., Lambalk, C.B., Schoemaker, J. and Vermes, I. (2000) Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil. Steril., 74, 245–250.[ISI][Medline]

Print, C.G. and Loveland, K.L. (2000) Germ cell suicide: new insights into apoptosis during spermatogenesis. Bioessays, 22, 423–430.[ISI][Medline]

Richburg, J.H. (2000) The relevance of spontaneous- and chemically-induced alterations in testicular germ cell apoptosis to toxicology. Toxicol. Lett., 112–113, 79–86.

Rodriguez, I., Ody, C., Araki, K., Garcia, I. and Vassalli, P. (1997) An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J., 16, 2262–2270.[Abstract/Free Full Text]

Sailer, B.L., Jost, L.K. and Evenson, D.P. (1995) Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J. Androl., 16, 80–87.[Abstract/Free Full Text]

Sakkas, D., Mariethoz, E. and St John, J.C. (1999a) Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp. Cell Res., 251, 350–355.[ISI][Medline]

Sakkas, D., Mariethoz, E., Manicardi, G., Bizzaro, D., Bianchi, P.G. and Bianchi, U. (1999b) Origin of DNA damage in ejaculated human spermatozoa. Rev. Reprod., 4, 31–37.[Abstract/Free Full Text]

Shen, H.M. and Ong, C.N. (2000) Detection of oxidative and damage in human sperm and its association with sperm function and male infertility. Free Radic. Biol. Med., 28, 529–536.[ISI][Medline]

Shiratsuchi, A., Umeda, M., Ohba, Y. and Nakanishi, Y. (1997) Recognition of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoli cells of the rat. J. Biol. Chem., 272, 2354–2358.[Abstract/Free Full Text]

Sinha Hikim, A.P., Wang, C., Lue, Y., Johnson, L., Wang, X.H. and Swerdloff, R.S. (1998) Spontaneous germ cell apoptosis in human: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death. J. Clin. Endocrinol. Metab., 83, 152–156.[Abstract/Free Full Text]

Sun, J.G., Jurisicova, A. and Casper, R.F. (1997) Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol. Reprod., 56, 602–607.[Abstract]

Tesarik, J., Greco, E., Cohen-Bacrie, P. and Mendoza, C. (1998) Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol. Hum. Reprod., 4, 757–762.[Abstract]

Vermes, I., Haanen, C., Steffens-Nakken, H. and Reutelingsperger, C. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods, 184, 39–51.[ISI][Medline]

Weil, M., Jacobson, M.D. and Raff, M.C. (1998) Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes. J. Cell Sci., 111, 2707–2715.[Abstract/Free Full Text]

Woolveridge, I. and Morris, I.D. (2000) Apoptosis in male reproductive toxicology. In Roberts R. (ed.) Apoptosis in toxicology. Taylor and Francis, New York/London, pp. 72–87.

World Health Organization (1999) WHO laboratory manual for the examination of human semen and semen–cervical mucus interaction, 4th edn. Cambridge University Press, Cambridge.

Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 284, 555–556.[ISI][Medline]

Submitted on May 10, 2001; resubmitted on August 20, 2001; accepted on January 8, 2002.