Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay

Liliana Ramos,1 and Alex M. M. Wetzels

Fertility Laboratory, Department of Obstetrics and Gynaecology, University Medical Centre St. Radboud, Geert Grooteplein 8, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In this study we present the physiological changes observed in ejaculated spermatozoa of normospermic men after exposure to hydrogen peroxide (H2O2) or {gamma} irradiation. METHODS: Motility changes as well as membrane and DNA-damage were determined in spermatozoa after incubation with 25 µmol/l of H2O2 during increasing intervals of time (0–60 min and after 24 h) or after irradiation of cells using {alpha} rays. Annexin V-binding in combination with propidium iodide was used for the assessment of membrane changes after each incubation time. TdT-mediated-dUTP nick-end labelling (TUNEL) was used to evaluate DNA damage. RESULTS: After 1 h incubation of the spermatozoa with H2O2, almost all cells were positive for Annexin-V, while no significantly increase in TUNEL positivity was observed. TUNEL results were significantly higher 24 h after incubation with H2O2 (10–16.3%, P = 0.03). In the control group (cumulus cells), an increase in the percentage of TUNEL positive cells was observed after 15 min of incubation with H2O2 and showed a five-fold increase after 24 h (from 8.1–72.1%, P < 0.001). TUNEL positive cells after {alpha} irradiation increased with the doses and post-irradiation time (from 10.8–47.2%). Interestingly, when only motile spermatozoa from irradiated samples were analysed, only 0.5% were TUNEL positive. CONCLUSION: Motility may be a relevant physiological marker for DNA-intact sperm after exposure of spermatozoa to H2O2 and {alpha} irradiation.

Key words: apoptosis/gamma radiation/ICSI/reactive oxygen species/TUNEL


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
With the introduction of intracytoplasmic sperm injection (ICSI), the risk of introducing DNA-damaged spermatozoa into the oocyte increases, especially in cases of poor sperm quality (Lopes et al.1998bGo; Twigg et al.1998Go; Ahmadi and Ng, 1999aGo; Irvine et al.2000Go). DNA-damage due to apoptosis has been found to occur in the testis during spermatogenesis predominately at spermatogonia and dividing cells level (Lee et al., 1997Go; Tesarik, et al., 1998Go; Yin et al., 1998Go; Host et al., 1999Go; Pentikainen et al., 1999Go; Sakkas et al., 1999Go; Print and Loveland, 2000Go). It is also increased in spermatozoa of poor quality as measured with the terminal deoxynucleotidyl transferase (TdT)-mediated UTP-nick end label (TUNEL) or Comet assay (Hughes et al., 1996Go; Sun et al., 1998; Irvine et al., 2000Go). The increased sensitivity to DNA damage in abnormal spermatozoa is probably due to failed chromatin condensation, which makes the DNA more accessible to damage (Bianchi et al., 1993Go; Sakkas et al., 1998Go; Twigg et al., 1998Go). Therefore, the presence of DNA fragmentation in ejaculated spermatozoa might correlate with defects in spermatogenesis, as has been suggested by Gandini and colleagues (Gandini et al., 2000Go). DNA fragmentation is more evident in atypical forms (Lopes et al., 1998bGo), confirming that morphology and sperm count correlate with testicular function. Elevated percentages of apoptotic spermatozoa have also been found after infections of the reproductive tract, cancer and other pathologies (Baccetti et al., 1996Go; Sharma et al., 1999Go; Gandini et al., 2000Go). However, scarce data have been published concerning the sensitivity of the cells to DNA damage or the mechanisms involved in cell death of ejaculated sperm from normospermic donors (Maione et al., 1997Go; Blanc-Layrac et al., 2000Go). This is important to elucidate especially in those pathologies where normal spermatozoa are exposed to non-physiological damaging agents. In vivo, this damage to the sperm cells probably occurs after completing maturation at the post-testicular level. This occurs for example in cases of anejaculation, where spermatozoa are exposed to elevated levels of reactive oxygen species (ROS) for long periods of time before (electro)ejaculation (Lamirande et al., 1995Go).

ROS are a known inductor of apoptosis in somatic cells (Ratan et al., 1994Go) and in maturing spermatozoa at the testicular level (Gorczyca et al., 1993Go). It is not clear whether ROS are also responsible for triggering DNA breaks in mature spermatozoa. Radiation is also responsible for chromosome aberrations (Tateno, et al., 1996Go) and DNA fragmentation in spermatozoa (Haines, et al., 1998Go).

The aim of this study was to determine and evaluate the physiological changes of ejaculated spermatozoa after exposure to free oxygen radicals or {gamma} radiation. The understanding of the possible mechanisms of cell death of ejaculated spermatozoa as a result of exposure to these agents may lead us to find markers for undamaged spermatozoa to use for ICSI. This might be important for the treatment of men whose semen has been stored for a long time in the epididymis, e.g. in cases of anejaculation or obstruction.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sperm samples and controls
Semen samples from normospermic donors attending our fertility department were used to study the effect of H2O2 (n = 7) or {gamma}-radiation (n = 4) on ejaculated spermatozoa (H2O2 was used as a source of free radicals). Samples were collected by masturbation into sterile containers and were delivered to the laboratory within 1 hour after ejaculation.

Sperm morphology was evaluated using the WHO criteria (WHO, 1999). Sperm concentration and motility assessment were performed in a Makler chamber after centrifuging the semen samples for 10 min at 500 g over an 80% PureSperm gradient (Nidacon International AB, Götenborg, Sweden). Subsequent washings were done with Human Tubal Fluid (HTF) culture medium (Quinn et al., 1985) supplemented with 10% plasma solution (CLB, Amsterdam, The Netherlands). The reason for using this fraction of the ejaculates is to have a homogeneous population to diminish variations between different aliquots of one sample.

Cumulus cells from six IVF patients were used as controls because, unlike spermatozoa, these (somatic) cells are transcriptionally active. Cumulus cells were collected and analysed on the day that oocytes were harvested for IVF.

Induction of DNA-strand breaks
Sperm samples were incubated in culture medium with a final concentration of 25 µmol/l H2O2 for different times (0, 5, 15, 30 and 60 min) at 30°C and cumulus cells were incubated for 0, 15 and 30 min at 37°C at the same H2O2 concentration. Higher concentrations of H2O2 caused immediate immobilization of spermatozoa (data not shown). Incubations were stopped by the addition of 10 µg/ml catalase (Sigma Chemicals, St Louis, MO, USA) and by washing the samples immediately after with IVF-medium. Samples that were incubated for 0 and 60 min were split into 2 aliquots to assess membrane and DNA-damage immediately, and after another 24 hours incubation (long term effect after damage induction). Control cells were incubated only for 0, 30 and 60 min and 24 h under similar experimental conditions (incubation at 37°C, reaction stopped by addition of 10 µg/ml catalase). Aliquots of sperm samples were also exposed to increasing doses of 0, 1, 5 and 50 Grays {gamma}-radiation (dose rate: 4.7 Grays/min) at room temperature (Gamma cell 1000, Atomic Energy of Canada, Canada). Motility changes, membrane and DNA-damage were assessed in all samples immediately after irradiation and again after 24 h incubation at 37°C.

Determination and selection of motile spermatozoa
Immediately after each incubation period with H2O2 or radiation, the percentage and quality of sperm motility was assessed in a Makler chamber to record any changes caused by H2O2 or {gamma}-radiation. For these purposes, any kind of motility observed, whether progressive or not, was considered `motile'.

The motile fraction from four out of seven sperm samples, which were previously irradiated with 5 Gray and incubated for 24 hours, was selected for the determination of DNA damage. The sperm suspension was diluted in 5% polyvinylpyrrolidone solution (PVP; Medicult a/s, Jylling, Denmark) and spermatozoa were individually selected using an ICSI-injection pipette (Humagen, Charlottesville, VA, USA) and micromanipulators. Motile sperm in PVP were transferred to a glass slide and labelled with TUNEL. At least 150 motile spermatozoa per sample were selected for analysis.

Determination of membrane damage
Membrane damage was assessed using Annexin-V stain (Nexins b.v., Kattendijke, The Netherlands) in combination with propidium iodide (PI, Sigma Chemicals) for evaluation of vitality (Glander and Schaller, 1999). In vital cells with an intact plasma membrane, phospholipid phosphatidylserine (PS) is located on the inner leaflet of the plasma membrane. The disturbance of membrane function starts with the translocation of PS from the inner to the outer leaflet and results in exposure of PS, where Annexin V binds. Samples were incubated for 15 min at room temperature in 0.5 µl of ready-to-use Annexin V-fluorescein isothiocyanate (FITC) solution in 300 µl binding buffer and mixed with 5 µl of PI. The FITC-labelled spermatozoa were analysed in a flow cytometer (Coulter Epics XL-MCL, Miami, FL, USA); a minimum of 10 000 cells was examined for each sample.

Determination of DNA-damage
DNA fragmentation induced in spermatozoa or cumulus cells was measured using the TUNEL assay (Cell Death Detection Kit, Roche Biochemicals, Mannheim, Germany) following the manufacturer's specifications with minor modifications. TUNEL identifies single and double stranded DNA breaks by labelling the free 3'-OH termini with modified nucleotides in an enzymatic reaction with terminal deoxynucleotidyl transferase (TdT). TdT polymerises free 3'-OH DNA ends in a template-independent manner, incorporating labelled nucleotides. Briefly, air-dried slides were fixed with 4% paraformaldehyde at room temperature and rinsed with phosphate-buffered saline (PBS), pH 7.4, and then permeabilised with 2% Triton X-100. The TdT-labelled nucleotide mix was added to each slide and incubated at 37°C for 60 min. Slides were rinsed twice in PBS and then counterstained with 10 mg/ml 4,6-diamidino-2-phenylindole (DAPI). From each sperm sample, one droplet of the sperm suspension was air dried onto a glass slide for the determination of DNA-breaks with the TUNEL assay. Controls were included in every experiment: for the negative control TdT was omitted in the nucleotide mix. Positive controls were generated by incubating the sperm cells for 10 min at room temperature with 50 units/ml DNase-I (Boehringer Mannheim, Mannheim, Germany). TUNEL labelling of the positive controls varied between 87–98% of cells.

At least 500 spermatozoa or 200 cumulus cells per sample were evaluated using a fluorescent microscope. The number of cells per field stained with DAPI (blue) were first counted; the number of cells with green fluorescence (TUNEL positive) were expressed as a percentage of the total sample.

Statistical analysis
Statistics were carried out with the statistical SPSS for Windows software package version 9.0 (SPSS Inc., Chicago, IL, USA). Calculations were performed using the paired two-tailed non-parametric test (Wilcoxon) or {chi}2 test where appropriate. Pearson's Correlation was used to calculate the correlation between TUNEL positive cells and radiation dose. Statistical differences were considered significant at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Effects of oxidative stress
Sperm motility decreased significantly after 5 minutes incubation with H2O2 (from 79 to 4%; P < 0.001), and was zero after 15 minutes. Annexin-V binding was found to increase shortly after the addition of H2O2 followed by an increase in PI positive cells. Annexin-V became almost 100% positive after just 60 min incubation (Figure 1Go). No significant increase of TUNEL positive cells was found within the first 60 min of incubation with H2O2, while a significant increase was found in those samples that were incubated for 24 h after damage induction with H2O2(10.0 versus 16.3% respectively; P = 0.03).



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Figure 1. Damage induced by H2O2 in spermatozoa after different intervals of time. Motility, membrane damage (Annexin-V and PI) and DNA fragmentation (TUNEL) were recorded after 0, 5, 15, 30 and 60 min incubation with 25 µmol/l H2O2 and again 24 h after damage induction (mean ± SD).

 
When control (cumulus) cells were exposed to similar incubation conditions (25 µmol/l H2O2, at 37°C), a significantly higher percentage of Annexin-V positive/PI negative and TUNEL positive cells was found after 30 minutes incubation (48.1 and 43.2% respectively). A five fold increase of TUNEL positive cells was found after an additional 24 h incubation (from 8.1 to 72.1%; P < 0.001), (see Figure 2Go).



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Figure 2. Damage induced by H2O2 in cumulus cells after 0, 15 and 30 minutes incubation with H2O2 and also 24 hours after damage induction. Results of the measurements of membrane damage (Annexin-V and PI) and DNA fragmentation (TUNEL) were significantly different after each interval of time (P < 0.05 – < 0.001; mean ± SD).

 
Effects of {gamma} radiation
DNA damage can be induced directly in spermatozoa by subjecting them to increasing doses of. {gamma} radiation. DNA-breaks (TUNEL positive cells measured immediately after irradiation) were found to increase proportionally (r = 0.91) to the radiation doses applied (9.4, 13.3, 14.3 and 25.2% for 0, 1, 5 and 50 Grays respectively, see Figure 3Go). Sperm motility decreased with the doses applied, although more than 50% of the cells were still motile even after 50 Gray. After incubation of the sperm samples for an additional 24 h, motility was further decreased and increasing numbers of TUNEL positive cells were observed (12.3, 19.2, 26.5 and 43.8% TUNEL positive cells after 0, 1, 5 and 50 Gray respectively; P < 0.05). All samples were also analysed for Annexin-V and PI binding and no increase in membrane damage was observed in these samples before or after irradiation (data not shown).



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Figure 3. (A) Percentage motile spermatozoa and (B) Percentage DNA fragmentation (TUNEL) observed in spermatozoa after applying increasing doses of gamma radiation. Light bars ({blacksquare}) represent the percentage motile cells (A) or TUNEL positive cells (B) immediately after radiation was applied; dark bars ({blacksquare}) represent the same samples 24 h later (mean + SD). Shared symbols ({square},*,**) represent significant differences (P < 0.01).

 
Selection of motile spermatozoa after {gamma} irradiation
The selected motile cells of four irradiated samples showed a negligible number of DNA breaks in the TUNEL assay. From the total of 350 individually selected motile cells, only two spermatozoa (0.5%) were positive for TUNEL. This is significantly different from the original sample from which the cells were selected (26.5%; P < 0.001).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, we analysed the changes that H2O2 or {gamma} irradiation caused in mature, ejaculated sperm cells from normospermic men at the membrane and nuclear levels. The data presented here provide evidence for the importance of sperm motility as a marker for the identification of intact DNA in spermatozoa. These results are based on DNA fragmentation analysis by the TUNEL assay after exposure of normal ejaculated spermatozoa to cytotoxic agents. Also, motility changes were observed after exposure of cells to {gamma} radiation, in correlation with the DNA damage induced, but independently of the membrane integrity as measured by Annexin V and PI. The damage observed showed a different pattern than expected.

Programmed cell death or apoptosis is the process by which cells activate an intrinsic death programme and kill themselves in a controlled way. Most, if not all, nucleated mammalian cells are capable of undergoing apoptosis and constitutively express all the proteins required to execute intrinsic cell death. The final stage of this programmed cell death is DNA fragmentation caused by activation of endogenous endonucleases. Fragmented DNA in spermatozoa can be detected with the Comet (Hughes et al., 1996Go; Donnelly et al., 2000Go) or the TUNEL assay (Sun et al., 1998). Both techniques are becoming widely used for the determination of DNA damage in spermatozoa of infertile patients (Hughes et al., 1996Go; Lopes et al., 1998aGo), in irradiated sperm cells (Haines et al., 1998Go) and in spermatozoa from patients exposed to cytotoxic agents during cancer treatment (Chatterjee et al., 2000Go).

To evaluate whether the changes found in spermatozoa, which lack an effective DNA-repair capacity and have no caspase activity (Weil et al., 1998Go), follow the normal pattern of cell death, cumulus cells were exposed to similar experimental conditions and analysed for membrane changes with the Annexin V-binding assay and for DNA-fragmentation with the TUNEL assay. Cumulus cells, which are transcriptionally active, became apoptotic after just 15 min exposure to H2O2, as can be observed from the measurements of Annexin V-binding and TUNEL. A significant increase in TUNEL positive cells was observed after 30 min incubation with H2O2 (P < 0.01). The difference observed between cumulus cells and spermatozoa after exposure to oxidative agents suggests that normal condensed chromatin in spermatozoa is more resistant to DNA fragmentation, probably due to its highly condensed nucleus and reduced nuclear activity.

Spermatozoa have high levels of polyunsaturated fatty acids in the membrane, which are responsible for fluidity and, therefore to the same extent, the motility of the sperm. The presence of free radicals causes changes in the distribution of the phosphatidyl serine (PS) of the membrane, which can be measured with the Annexin V-binding assay (Glander and Schaller, 1999). Motility strongly decreased as soon as 1 min after the addition of H2O2 (data not shown), and all spermatozoa became immotile after 15 min. Although changes at the membrane level are evident, no changes in the percentage of TUNEL positive cells were found during the first hour of incubation in the presence of H2O2, confirming the results of Hughes et al., (1996), but in contrast to the results presented by Lopes et al., (1998a). This difference can be explained by the fact that the last author used samples from infertile men, which are probably more sensitive to DNA damage than spermatozoa from normal men. Further, higher levels of H2O2 were used by these authors. In our setting, it was only when the sperm were incubated for an additional 24 h after damage induction that a significant increase in DNA-damage was detected compared with the samples without previous damage induction (16.3 versus 10.0 % respectively; P < 0.05). This result suggests that normal spermatozoa in the presence of oxidative agents become immotile long before any DNA-damage can be detected.

DNA breaks in spermatozoa can be directly induced by irradiation with {gamma}-rays (Haines et al.1998Go). However, fertilisation capacity and pronuclear formation is not impaired in damaged cells (Twigg et al., 1998Go; Ahmadi and Ng, 1999aGo, bGo). We also found a significant increase in DNA fragmentation, which is proportional to the doses of radiation applied. However there was neither a significant difference in motility, nor a change in the Annexin V-binding capacity, immediately after irradiation of the cells. This suggests that the damage at first instance seems to be restricted to the nucleus. Interestingly, DNA damage was more evident after incubation of the cells for 24 h after the radiation was applied, with a significant decrease in motility (P = 0.01) and an increase in TUNEL positivity (P = 0.03).

The activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA has been already reported (Gorczyca et al., 1993Go; Weil et al., 1998Go). We hypothesise that the initial damage is enhanced by the activation of an intrinsic mechanism (activation of endonucleases) that causes the cells to go into apoptosis. Although mature spermatozoa are not transcriptionally active, endonuclease activity is still found in both epididymal and ejaculated spermatozoa and it can be triggered in response to exogenous DNA, causing partial degradation of the sperm endogenous chromosomal DNA (Maione et al., 1997Go; Spadafora, 1998Go). Therefore, in cases of obstructive azoospermia or anejaculation, cell breakdown caused either by cell ageing or ROS can contribute large amounts of DNA fragments, which may be responsible for the activation of endogenous endonucleases. It is also possible that mitochondria, which not only metabolise energy for sperm flagellar propulsion but also play an important role in cell death, are affected by the presence of a high concentration of ROS, disturbing the membranes of the organelle and its metabolising function. Dysfunctional mitochondria (Donnelly et al., 2000Go) together with other mechanisms such as oxidation of glycolitic (cytosolic) enzymes may also contribute to the changes of motility observed in spermatozoa.

Using motile sperm selected from sperm samples with high levels of DNA-breaks (from the 5 Gray {gamma}-irradiated aliquots, mean TUNEL 26.5%), we found only 2 out of 350 cells (0.5%) to be positive in the TUNEL assay (P < 0.001). This finding indicates that motility can be used as one of the physiological markers for the selection of undamaged cells in ICSI procedures. This is a very important fact, because damaged spermatozoa still have the ability to fertilise the oocytes after ICSI (Twigg et al., 1998Go), but lead to poor embryo development and high pregnancy loss rates (Sakkas et al., 1998Go; Ahmadi and Ng, 1999bGo).

In conclusion, the results presented in this study may be relevant for the understanding of the processes at both membrane and DNA level which are involved in sperm damage following their exposure to agents such as H2O2 or {gamma} irradiation. Motility was found to be a relevant physiological marker for intact DNA. Further research on DNA damage of the motile (morphologically normal) spermatozoa which are the candidates to be selected for ICSI, should be carried out in order to evaluate the reliability of motility as a marker for DNA integrity.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors would like to thank Mr.A.Pennings for his technical assistance with FACS measurements. This research was supported by the Ministry of Health, Welfare and Sport (VWS) of the Netherlands.


    Notes
 
1 To whom correspondence should be addressed. E-mail: l.ramos{at}obgyn.azn.nl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 4, 2000; accepted on April 30, 2001.