Incidence of Fas positivity and deoxyribonucleic ... - Fertility and Sterility

FERTILITY AND STERILITY威 VOL. 81, SUPPL. 1, MARCH 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.

Incidence of Fas positivity and deoxyribonucleic acid double-stranded breaks in human ejaculated sperm Carmel M. McVicar, Ph.D.,a Neil McClure, F.R.C.O.G.,a Kate Williamson, Ph.D.,b Lauren H. Dalzell, B.Sc.,a and Sheena E. M. Lewis, Ph.D.a School of Medicine, Queens University Belfast, Institute of Clinical Science, Belfast, N. Ireland

Received March 14, 2003; revised and accepted October 20, 2003. Supported by The Research and Development Office, Northern Ireland, through the Recognised Research Group programme 5.9. Presented at the 18th Annual Meeting of the European Society of Human Reproduction, Vienna, Austria, June 28 – July 3, 2002. Reprint requests: Carmel M. McVicar, Ph.D., Department of Obstetrics and Gynaecology, Queens University Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, Ireland (FAX: 44-28-90328247; E-mail: [email protected]). a Department of Obstetrics and Gynaecology. b Department of Pathology. 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2003. 10.013

Objective: To determine the incidence of Fas positivity and DNA double-strand breaks (DSB) as indicators of early- and late-stage apoptosis in ejaculated sperm. Design: Fas positivity was assessed by flow cytometry and DSB by the neutral Comet assay. Setting: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. Patient(s) and Intervention(s): Forty-five infertile men undergoing infertility investigations and 10 fertile men undergoing vasectomies. Main Outcome Measure(s): Percentage Fas-positive cells, percentage DNA fragmentation, olive tail moment. Result(s): The apoptotic marker Fas was detected in ejaculated sperm, with a higher incidence of Fas positivity in teratozoospermic and asthenozoospermic than in normozospermic semen. No Fas positivity was observed in fertile mens’ sperm. Deoxyribonucleic acid fragmentation (DSB) was greater in infertile than in fertile men’s sperm and also greater in sperm in semen than in sperm prepared for assisted conception. There was an inverse relationship between DSB and both sperm concentration and motility. There was no relationship between Fas positivity and DNA damage. Conclusion(s): Fas was expressed in sperm of infertile men. In contrast, DNA fragmentation was observed in all sperm of fertile and infertile men and correlated with inadequate concentration and motility, which suggests that sperm DSB are ubiquitous and are not solely associated with apoptosis. (Fertil Steril威 2004; 81(Suppl 1):767–74. ©2004 by American Society for Reproductive Medicine.) Key Words: DNA double-stranded breaks, ejaculated sperm, Fas, flow cytometry, neutral Comet assay

Unlike other mammalian species, only 14% of sperm need to display normal morphology for a human semen sample to be classified as normal by the Tygerberg strict criteria (1). The reason such a high level of abnormality can be seen in so-called “normal” ejaculate remains unknown. One suggestion is that although these abnormal sperm were destined for apoptosis they were released from the testis before the process was completed (2). Apoptosis plays an important role in the testis by controlling germ cell number (3, 4). Fifty–70% of germ cells ultimately undergo apoptosis at different stages of spermatogenesis (5–7). The process is very specific and is restricted to the germ cells of the seminiferous tubules: somatic (Leydig and Sertoli) cells show no signs of degeneration (5, 8).

Apoptosis can be initiated through deathreceptor ligation, p53 upregulation, or other diverse mechanisms. Apoptosis can be regulated by many protein interactions, including those of the Bcl-2 family, before the cells commit to the process via the caspases and endonucleases and results in the final structural changes of DNA fragmentation and blebbing. To assess the proportion of sperm primed for apoptosis, we assessed the levels of the apoptotic marker Fas, because it is believed to be a key initiator in the testis (3). As an indicator of late-stage apoptosis we measured the incidence of DNA double-stranded breaks (DSB) (9). We also determined the association between Fas expression and DSB within sperm samples. Unlike single-strand breaks (SSB, measured typically by the alkaline Comet or 767

Tdt-mediated dUTP-biotin nick end labeling [TUNEL] assays), which might occur randomly and might be more easily repaired after fertilization (10), DSB of DNA are a classic sign of apoptotic execution. In addition, we have compared the incidence of the apoptotic marker Fas on subpopulations of normal and abnormal sperm from both fertile and infertile men and tested for a relationship between Fas expression and the classic sperm parameters (i.e., concentration, motility, and morphology).

MATERIALS AND METHODS Collection of Semen Samples Semen samples were obtained from men attending for infertility investigations (n ⫽ 45) who had been sexually abstinent for a recommended 2–5 days. Semen profiles were classified as teratozoospermic (n ⫽ 6), oligoasthenoteratozoospermic (n ⫽ 14), oligoteratozoospermic (n ⫽ 1), oligoasthenozoospermic (n ⫽ 1), asthenoteratozoospermic (n ⫽ 12) or normozoospermic (n ⫽ 9) according to the World Health Organization 1999 criteria. All subjects were the partners of women who had not conceived after 2 years of unprotected intercourse. Fertile semen samples (n ⫽ 10) were obtained from men before vasectomy and again after 2–5 days of sexual abstinence. Written consent for participation was obtained, and the project was approved by the Queen’s University Belfast Research and Ethics Committee. In all cases a conventional light microscopic semen analysis was performed within 1 hour of sample production to determine sperm concentration, motility, and morphology. An aliquot of semen (⬃500 ␮L) was retained for further experiments; the remainder was prepared by density centrifugation.

Preparation of Samples Samples were prepared with a two-step, discontinuous, Percoll gradient (97.0%– 47.5%; Pharmacia Biotech AB, Uppsala, Sweden). Each aliquot of liquefied semen was layered on top of the gradient and centrifuged at 450g for 12 minutes. The resulting sperm pellet was concentrated by centrifugation at 200g for 6 minutes. The final sperm preparation was suspended in 200 ␮l of Biggers, Whitten, and Whittingham medium (BWW) (11).

Sperm Motility Parameters

Semen samples were analyzed with 20-␮m counting chambers (Microcell; Conception Technologies, La Jolla, CA). Sperm motility parameters were measured at 37°C (Hamilton-Thorne Integrated Visual Optical System (IVOS) Sperm Analyzer Version 10.7; Hamilton-Thorne Research, Beverley, MA), as described previously (12).

Sperm Morphology The Tygerberg strict criteria (1) were applied to assess sperm morphology in the samples examined, as previously reported (12). 768

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Fas and DNA strand breaks in sperm

DNA Fragmentation (DSB) A novel modified neutral single-cell gel electrophoresis (Comet) assay was developed from the alkaline Comet assay (12) and the neutral Comet assay (13). Fully frosted microscope slides (Surgipath Europe, Peterborough, United Kingdom) were gently heated, covered with 100 ␮l of 0.5% normal melting point agarose in Ca2⫹ and Mg2⫹ free phosphate-buffered saline (PBS; Sigma, Poole, United Kingdom) at 45°C and immediately covered with a large (22 ⫻ 50 mm2) coverslip. Slides were placed in a chilled metal tray and left at 4°C for 20 min to allow the agarose to solidify. Coverslips were then removed, and 6 ⫻ 106 sperm in 10 ␮l BWW were mixed with 75 ␮l of 0.5% low melting point agarose at 37°C. This cell suspension was rapidly pipetted on top of the first agarose layer, covered with a coverslip, and allowed to solidify at 4°C for 15 min. The cells were lysed by removal of the coverslips and immersion of the slides for 1 hour at 4°C in a Coplin jar containing 2.5 mol/L NaCl, 100 mmol/L Na2 ethylenediaminetetraacetic acid (EDTA), and 10 mmol/L Tris (pH 10), with 2% Triton X-100 (Sigma, United Kingdom) added just before use. Slides were incubated for 30 minutes at 4°C with 10 mmol/L dithiothreitol (Sigma, United Kingdom), followed by 90 minutes incubation at 20°C with 4 mmol/L lithium diiodosalicyclate (Sigma, United Kingdom) (14) to allow the DNA to decondense. The slides were removed from the lysis solution and drained of any remaining fluid. Slides were placed into a horizontal gel electrophoresis tank (11.14; Anachem, Luton, Bedfordshire, United Kingdom) filled with fresh neutral electrophoresis solution (Tris-borate-EDTA buffer [90 mmol/L Tris, 90 mmol/L boric acid, and 2 mmol/L EDTA], pH7) and allowed to equilibrate for 20 minutes at room temperature before electrophoresis. The DSB were separated by electrophoresis for 45 minutes at 25 V adjusted to 19 mA by the raising or lowering of the buffer level in the tank. After electrophoresis the slides were drained and flooded with three changes of neutralization buffer (0.4 mol/L Tris, pH 7; Sigma, United Kingdom) for 5 min each. The slides were drained and stained with ethidium bromide staining (12) before being viewed with an epifluoresence microscope (Nikon E600; Nikon, Tokyo, Japan). Intact and damaged DNA were analyzed with an image analysis system and specialized software (Komet 3.1; Kinetic Imaging, Nottingham, United Kingdom) (12). The percentage head and tail DNA was measured in 100 sperm from each sample (12). In contrast to the percentage DNA damage, which is a percentage measure of the amount of fragmented DNA only, the olive tail movement (OTM) measures both the percentage DNA and the length of the damaged tail drawn out by electrophoresis. Olive tail moment is the percentage of fragmented DNA in the tail times the tail length. This is the conventional method used to measure doubleVol. 81, Suppl 1, March 2004

strand damage (15). Sperm with OTM ⬎10 were classified as apoptotic. The alkaline Comet assay was used as previously described (12). The method was the same as the neutral Comet assay until the stage that the cells were lysed. The cells were lysed by removal of the coverslips and immersion of the slides for 1 hour at 4°C in a Coplin jar containing 2.5 mol/L NaCl, 100 mmol/L Na2 EDTA, and 10 mmol/L Tris (pH 10), with 1% Triton X-100 (Sigma, United Kingdom) added just before use. Slides were incubated for 30 minutes at 4°C with 10 mmol/L dithiothreitol (Sigma, United Kingdom), followed by a 90-minute incubation at 20°C with 4 mmol/L lithium diiodosalicyclate (Sigma, United Kingdom) (14) to allow the DNA to decondense. The slides were removed from the lysis solution and drained of any remaining fluid. Slides were placed into a horizontal gel electrophoresis tank filled with fresh alkaline electrophoresis solution (Tris-borate-EDTA buffer [90 mmol/L Tris, 90 mmol/L boric acid, and 2 mol/L EDTA], pH7) and allowed to equilibrate for 20 min at room temperature before electrophoresis. The double- and single-strand breaks were separated by electrophoresis for 10 min at 25 V (0.714 V/cm) adjusted to 300 mA by the raising or lowering of the buffer level in the tank. After electrophoresis the slides were drained and flooded with three changes of neutralization buffer (0.4 mol/L Tris, pH 7.5; Sigma, United Kingdom) for 5 min each. The slides were drained and stained with ethidium bromide staining (12) before being viewed with an epifluoresence microscope (Nikon E600). Intact and damaged DNA was analyzed with an image analysis system and specialized software (Komet 3.1) (12).

Location of Fas on Sperm by Immunocytochemistry

Sperm (3 ⫻ 106) were aliquoted to four tubes, washed in 4 ml of BWW (11), and centrifuged for 10 min at 1,500 ⫻ g. Fifty microliters of sperm pellets from each sample were resuspended in 500 ␮l of 37°C KMT medium (100 mmol/L KCl, 2 mmol/L MgCl2, 10 mmol/L Tris-HCl; pH 7) (16) on the poly-L-lysine– coated microscopy coverslips (Sigma, United Kingdom) and allowed to attach for 5 minutes on a slide warmer. One hundred microliters of 8 ␮g/mL of CD95 mouse antihuman antibody (Beckman Coulter, High Wycombe, United Kingdom) diluted in Triton X-100 (2.5 mL; BDH Laboratory Supplies, Poole, United Kingdom), sodium azide (0.5 g; Sigma, St. Louis, MO), and bovine serum albumin (0.5 g; Sigma, St. Louis, MO) were dissolved in 0.1 mol/L PBS (500 mL; Sigma, St. Louis, MO) was added to duplicate slides. As controls, 100 ␮l of isotype control immunoglobulin (Ig)G1 was added to one slide and no primary antibody to a second. All slides were incubated for 2 hours at 37°C. The unbound primary antibody was removed with PBS (pH 7) wash before incubation of the sperm with 100 ␮l of secondFERTILITY & STERILITY威

FIGURE 1 Fluorescence micrograph displaying Fas positivity on sperm. Fas immunostaining over the head and the midpiece of some abnormally shaped sperm in a teratozoospermic sample. Original magnification, ⫻1,000.

McVicar. Fas and DNA strand breaks in sperm. Fertil Steril 2004.

ary fluorescein isothiocyanate conjugate (FITC)-conjugated goat antimouse (Beckman Coulter) diluted 1:50 in antibody diluent for 1 hour at 37°C. Cells were washed once with PBS and submerged in 1% paraformaldehyde fixative for 40 minutes. The cells were washed again in 0.01 mol/L PBS, mounted in PBS/glycerol, and viewed and photographed with a confocal scanning laser microscope fitted with a supplementary EY 455-nm excitation filter (Bio-Rad MRC 500; Bio-Rad Laboratories, Hercules, CA). The same settings were used to obtain the experimental and negative control images.

Incidence of Fas Positivity Semen samples containing contaminant cells, such as neutrophils, were sent for bacteriologic analysis and omitted from this study. Aliquots containing 1 ⫻ 106 sperm were pipetted into four test tubes, washed in 4 mL of BWW (11), and centrifuged for 10 min at 1500g. Forty microliters of 20 ␮g/mL of CD95 mouse antihuman antibody (Beckman Coulter) in antibody diluent were added to triplicate tubes; 40 ␮l of isotype control IgG1 were added to the control tube before incubation for 2 hours at 37°C. The unbound primary antibody was removed with PBS (pH 7) before sperm were incubated with the secondary antibody and fixed as described for immunocytochemistry. Sperm were stored in the dark at 4°C and analyzed with a flow cytometer (EPICS ELITE; Coulter) within 24 hours. The percentage of positive cells was determined by subtraction of the isotype from the test histograms with specialized software (Immuno 4; Beckman Coulter) (Fig. 1). 769

Western Blot Analysis

Each patient’s sample was used at a concentration of 5 ⫻ 10 (n ⫽ 4). The following enzymes were added to the samples: 10 mmol/L E64; 1 mmol/L leupeptin; 10 mmol/L pepstatin; 4 ␮mol/L pefbloc, and 1.3 mmol/L EDTA. A eukaryotic protein extraction kit (Mem–PER; Pierce Biotechnology, Rockford, IL; cat. no. 89826) was used to extract the hydrophilic phase from the hydrophobic layer, which contains the membrane proteins. The samples were transferred to 4%–12% polyacrylamide gel. The proteins were transferred to blotting paper by electrophoresis at room temperature in transfer buffer NuPAGE (25 mmol/L bicine, 25 mmol/L Bis-Tris [free base], 1 mmol/L EDTA, and 0.05 mmol/L chlorobutanol) at 30 V for 1 hour (Xcell Surelock Mini-Cell; Invitrogen, Paisley, United Kingdom; cat. no. 9003). Western blotting was performed with the rabbit polyclonal antibody to human Fas (sc-715/C-20; Santa Cruz Biotechnology, Santa Cruz, CA) at 0.1 ␮g/mL in TBS-T (20 mmol/L Tris [pH 7.5], 137 mmol/L NaCl, and 0.01% Tween 20) overnight at room temperature on the rocker, followed by alkaline phosphatase (1/1,000) goat antirabbit (Sigma, United Kingdom; cat. no. A0418).

FIGURE 2

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Relationship between Fas immunofluorescence in fertile and infertile men’s sperm as assessed by flow cytometry. This graph compares the incidence of Fas positivity in fertile (n ⫽ 6) and infertile (n ⫽ 24) men’s sperm. There was a significantly higher incidence of Fas positivity in infertile than in fertile men’s sperm (P⬍.05, t-test).

Female Investigations

The female partners of the infertile men (n ⫽ 24) whose sperm displayed Fas positivity were reviewed for ovulation and tubal occlusions. Those patients were then allocated into two groups: either a male factor or a female factor.

Statistical Analysis Data were analyzed with special software (SPSS 10 for Windows; SPSS, Chicago, IL). To take into consideration the non-Gaussian distribution of data, the nonparametric Wilcoxon signed rank test was used to determine the relationship between the percentage of cells with fragmented DNA in semen and prepared sperm from the same ejaculate. The percentage of Fas-positive sperm was converted to unstandarized residuals owing to the high proportion of zero values. Pearson’s correlation with the two-tailed test of significance was used to assess the differences between the Fas-positive sperm, OTM, and the abnormal sperm parameters. The percentages of Fas positivity in fertile and infertile patients were compared with Student’s t-test.

RESULTS Determination of Fas Immunofluorescence in Sperm Intense Fas-immunoreactivity was observed at the nuclear region and the midpiece of abnormally shaped cells (Fig. 1). There was no evidence of Fas immunofluorescence in morphologically normal sperm. The negative control (IgG1 isotype) displayed no immunoreactivity; thus the Fas antibody used in this study was specific on the sperm assessed. No Fas immunostaining was obtained in fertile patients. 770

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Relationship Between Fas Immunofluorescence Staining in Sperm from Fertile and Infertile Men, Assessed by Flow Cytometry A marked difference in Fas immunofluorescence, as determined by flow cytometry, was observed between fertile and infertile men’s sperm with none of the fertile men’s sperm displayed Fas positivity, and the infertile patients’ sperm displayed a range between 0.0% and 55.0%. Of those patients whose sperm were Fas positive, 96% were diagnosed as having male infertility only, because all female partners were ovulating regularly, and no tubal occlusions had been identified (Fig. 2).

Comparison of DNA Fragmentation (DSB and SSB) in Semen and Prepared Sperm from Infertile and Fertile men with the Neutral (DSB) and Alkaline Comet Assay (DSB and SSB) In the infertile men, there was significantly less fragmented DNA (DSB and SSB) in prepared sperm than in semen, as assessed by the alkaline Comet assay (P⬍.05). This was also true of DNA (DSB only), as determined by the Vol. 81, Suppl 1, March 2004

neutral Comet assay (DSB) (P⬍.05). The substantially lower mean levels of fragmented DNA (16.0% ⫾ 1.3%; P⬍.005) found with the neutral (DSB) compared with the alkaline Comet assay (38.0% ⫾ 2.5; DSB and SSB) in semen indicated that approximately 22.0% of DNA damage was composed of single- rather than double-strand breaks in sperm. In the infertile men, the lower mean levels (10.0% ⫾ 0.9%; P⬍.005) of fragmented DNA found with the neutral (DSB) compared with the alkaline Comet assay (27.0% ⫾ 2.1%; DSB and SSB) indicated that approximately 17.0% of DNA damage was composed of single- rather than doublestrand breaks in prepared sperm. In the fertile men, there was less fragmented DNA (DSB and SSB) in prepared sperm than in semen, as assessed by the alkaline Comet assay. However, this trend did not achieve significance (P⫽.07). In contrast, a significant difference for DSB only was detected by the neutral Comet assay (P⬍.05). The substantially lower mean levels (10.0% ⫾ 1.0%; P⬍.05) of fragmented DNA found with the neutral (DSB) than with the alkaline Comet assay (26.6% ⫾ 5.3%; DSB and SSB) indicated that DNA damage is composed of approximately 17.0% single-strand breaks in sperm. In the fertile men’s prepared samples, the lower mean levels (7.0% ⫾ 0.7%) of fragmented DNA (DSB) found with the neutral compared with the alkaline Comet assay (19.0% ⫾ 5.2%; DSB and SSB) indicated that DNA damage was composed of approximately 12.0% single- rather than double-strand breaks in prepared sperm. However, this difference was not statistically significant (P⫽.20).

DNA Fragmentation as Defined by OTM Significantly greater OTMs were observed in semen compared with prepared sperm (P⬍.005). Of infertile patients, 93% had an OTM ⬎10 in semen compared with 30% in prepared sperm. Assessment of OTM in semen and prepared sperm of fertile men showed that 40% had an OTM ⬎10 in semen compared with 30% in prepared sperm, but this was not significantly different P⫽.53.

Relationship Between Percent Fas Positivity and DNA Damage in Sperm There was no relationship between the samples that expressed Fas positivity and the DSB (OTM ⬎10) (r ⫽ 0.14, P⫽.45, Pearson’s correlation; Fig. 3).

FIGURE 3 Relationship between percent Fas positivity and DNA damage in sperm. There was no relationship between the samples that expressed Fas positivity (columns) and the DSB (OTM ⬎10) (line) (r ⫽ 0.14, P⫽.45, Pearson’s correlation). Teratozoospermic, n ⫽ 24; normozoospermic, n ⫽ 6.


probit values of OTM (r ⫽ ⫺0.40, P⬍.05 and r ⫽ ⫺0.39, P⬍.05, respectively, Pearson’s correlation). No significant differences were observed between the OTM and morphology (r ⫽ 0.13, P⫽.51, Pearson’s correlation).

Western Blot Analysis With a Western blot, Fas was detected (a single band at 48 kDa) in sperm from infertile men (n ⫽ 3) but not in sperm from a fertile control (data not shown).

FIGURE 4 Relationship between Fas and morphology. There was an inverse relationship between sperm morphology (columns) and the percentage of Fas-positive cells (line) (r ⫽ ⫺0.40, P⬍.05, Pearson’s correlation between the Fas positivity of teratozoospermic and normozoospermic samples according to their probit values). Teratozoospermic, n ⫽ 24; normozoospermic, n ⫽ 6.

Relationship Between Fas, OTM, and Classic Sperm Parameters There was no relationship between the percentage of Fas-positive sperm and sperm concentration (r ⫽ .13, P⫽.51, Pearson’s correlation). There was an inverse relationship between both sperm morphology (Fig. 4) and motility (Fig. 5) and the probit values for percentage of Faspositive cells (r ⫽ ⫺0.40, P⬍.05, Pearson’s correlation). There was an inverse relationship between OTM ⬎10 and concentration and motility of the sample as assessed by the FERTILITY & STERILITY威


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FIGURE 5 Relationship between Fas and motility. There was an inverse relationship between sperm motility (columns) and the percentage of Fas-positive cells (line) (r ⫽ ⫺0.40, P⬍.05, Pearson’s correlation between the Fas positivity of asthenozoospermic and normozoospermic samples according to their probit values). Teratozoospermic, n ⫽ 24; normozoospermic, n ⫽ 6.


DISCUSSION Until recently it has been accepted that a mature sperm’s inability to synthesize new proteins deemed it impossible for sperm to respond to apoptotic signals. However, there are now reports in the literature that challenge our understanding of the role of apoptosis in sperm. The detection of Fas on ejaculated sperm (17), the high proportion of sperm with potentially apoptotic mitochondria in subpopulations of poor-quality sperm (12), evidence of RNA (18, 19), and the presence of endonuclease activity (20) in mature postspermiogenetic sperm all support the possibility of on-going apoptosis. Fas-mediated apoptosis has been observed in enucleated cells, thus all of the components necessary for apoptotic signal transduction through to cell death are present without DNA degradation (21). Although transcription and translation are minimal in sperm, it has been shown that it is essential to inhibit protein synthesis to suppress the survival pathways to trigger cell death in HeLa cells (22). These findings, together with our inability to explain the enormous proportion of abnormal sperm even in the semen of fertile men, lead to the hypothesis that apoptosis could be executed in sperm without input from nuclear DNA. In this study, no sperm from fertile men displayed Fas positivity, whereas 70% of infertile samples were Fas positive. Of the Fas-positive samples, 96% of those couples had male infertility only, with no abnormalities detected in the female partner. This suggests a strong relationship between Fas signaling of sperm and the men’s infertility. On comparing Fas marking with classic semen parameters we found a negative relationship with morphology and motility. This suggests that Fas expression could be a useful addition to a semen analysis and supports the evidence (2) that apoptosis 772

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is a means of eliminating defective sperm. However, unlike Sakkas et al. (17), we observed no relationship between the percentage Fas-positive sperm and the concentration of the sample. As Sertoli cells normally activate the Fas system as a means of limiting sperm number (3, 4), it would be surprising if they initiated the apoptotic cascade when spermatogenesis was already defective. One reason for the discrepancy between this study and that of Sakkas et al. might be a methodologic difference. If the antibody became saturated at high sperm concentrations it might appear that there was a higher incidence of Fas marking in samples of low concentration. To prevent this in our study, a constant ratio of sperm (1 ⫻ 106) to antibody was used for each test so that the antibody antigen reaction was never saturated. In addition, the relevant isotype IgG1 for the Fas antibody was added as a negative control instead of the secondary FITC antibody only to detect any nonspecific activity. The Fas system is one of the key apoptosis-related systems in the development of human testicular germ cells (23). Sertoli cells express FasL, which binds to Fas on Faspositive germ cells, thus initiating apoptosis and limiting the size of the germ cell population to numbers that can be supported (3, 4). Fas ligation induces the trimerization of the Fas receptor, which activates caspase 8 (FLICE/MACH) through an adaptor Fas-associating protein with death domain (FADD)/MORT1 (24). The signal is then amplified by caspase 8 cleavage of Bid, a proapoptotic marker, generating a C-terminal fragment of the protein that can bind to mitochondria, thus triggering cytochrome C release that is followed by activation of the effector caspases and subsequent cell death (25). However, caspase 8 has also been demonstrated to be activated independently of Fas ligation (26 –28). To determine if Fas priming was associated with irreversible apoptosis we also measured DNA fragmentation in these sperm. Numerous previous studies have used TUNEL assays to focus on DNA damage as indicative of apoptosis (29 –32). However, the TUNEL assay detects DNA breaks through labeling of 3⬘-OH termini with terminal deoxynucleotidyl transferase and reacts preferentially with either singlestranded DNA molecules or double-stranded DNA with 3⬘ overhangs. Such aberrations in DNA structure are more likely to be due to random oxidative damage than to orchestrated and controlled programmed cell death. The classic DNA measure of apoptosis is “laddering,” whereby gel electrophoresis can demonstrate nucleosomal-sized fragments of approximately 180 base pairs. However, the unique protamine packaging of sperm DNA renders this technique inapplicable. To overcome this problem we used a modified neutral Comet assay that detects only DSB of DNA. As well as its potential as an apoptotic marker, DSB damage is possibly more important in relation to fertility, because less than 8% of sperm DNA damage can be repaired within the oocyte Vol. 81, Suppl 1, March 2004

(10, 32), and DSB are more difficult to repair than SSB. When we compared total fragmentation (i.e., SSB and DSB) using the standard alkaline Comet assay we found that just 16% of DNA damage was due to DSB in semen. As shown previously with the alkaline Comet assay (12), sperm preparation isolates a subpopulation with less DNA fragmentation (DSB), thus confirming again its efficacy in artificial reproductive technology. We have also demonstrated that fertile men have substantially fewer DSB in their sperm (10% ⫾ 0.96%) relative to infertile men (16.0% ⫾ 1.3%). Nonetheless, in sharp contrast to somatic cells, in which damage is exceptional (33) rather than normal, in our experience virtually all sperm have at least low levels of DNA damage (34). These observations make it highly unlikely that all DNA damage is apoptotic, thus the damage must have a different origin. Using the sperm chromatin structure (SCSA) assay, Evenson’s group has shown (35) that when the denatured DNA was above a threshold (⬎30%), ultimately fertile couples took longer to conceive. This SCSA data also predicted 39% of the miscarriages that subsequently occurred within the group. Also, in a large group of Danish first-pregnancy planners, fecundity was shown to decline (36) as a function of the percentage of sperm with abnormal chromatin, and no pregnancies resulted after assisted reproductive techniques if ⱖ27% of sperm in the neat sample showed DNA denaturation by SCSA (37). Whether the low levels of basal damage observed in fertile men are of any major consequence to their fertility is debatable, because a high proportion of DNA is made up of noncoding, intron sequences and is therefore unimportant for gene transmission. Here we found that double-stranded DNA fragmentation (DSB) was a useful indicator of sperm quality and inversely related to concentration (r ⫽ ⫺0.40), thus supporting existing data that sperm from infertile men have more DNA damage than do those of fertile men (32, 37–39). We found that there was no relationship between the samples displaying Fas positivity and the DSB (r ⫽ 0.14, P⫽.45). This suggests that the signal marking of sperm for apoptosis in the testis is not associated with execution of the process. Patients with complete spermiogenic failure have been reported to display high levels of apoptotic germ cells (40). Biopsies of their testicular tissue cultured in vitro resulted in successful pregnancies. Our data support those of Sakkas et al. (41), who also observed that apoptotic markers and DNA damage did not necessarily exist in unison. This study suggests that the DNA damage that is ubiquitous in sperm might be due to other factors, such as reactive oxygen species (ROS) (36, 42). However, there was a greater incidence of Fas positivity in infertile semen from men. Because Fas upregulation is an external apoptotic trigger, this might indicate that infertile men’s sperm have been in a more “hostile” environment than fertile men’s sperm and that the infertile men’s sperm have sustained subapoptotic FERTILITY & STERILITY威

damage. It is not possible to speculate whether the apoptotic process itself is defective in infertile men’s sperm or whether the trigger has occurred at a time when apoptosis has not been possible.

Acknowledgments: The authors thank Mrs. Margaret Kennedy, HNC, for her preparation of sperm samples, Mr. Ken Arthur, MMedSci, F.I.B.M.S., for assistance with flow cytometry, Dr. David Crockard, Ph.D., for supplying neutrophils, Dr. Fionnuala Lundy, Ph.D., for helping with Western blotting, and Mrs. Kathy Pogue, B.Sc., and Dr. Perry Maxwell, Ph.D., for their expertise with immunocytochemistry. The authors also thank Mr. Mike Stevenson, B.Sc., for his help with statistics.

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Vol. 81, Suppl 1, March 2004