Mol. Hum. Reprod. Advance Access originally published online on June 21, 2007
Molecular Human Reproduction 2007 13(9):621-631; doi:10.1093/molehr/gam046
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Characterization of M540 bodies in human semen: evidence that they are apoptotic bodies
1Andrology Unit, Department of Clinical Physiopathology, Center for Research, Transfer and High Education, DENOthe, University of Florence, Viale Pieraccini 6, I-50139 Firenze, Italy 2Department of Anatomy Histology and Forensic Medicine, University of Florence, I-50134 Florence, Italy
3 Correspondence address. Tel: +39 055 427 1486; Fax: +39 055 427 1371; E-mail: e.baldi{at}dfc.unifi.it, m.muratori{at}dfc.unifi.it
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Abstract |
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Our group has recently identified, in human semen, round bodies of different size and density which were termed M540 bodies due to their staining with the fluorochrome merocyanine 540. Here, we investigate the hypothesis that such structures represent apoptotic bodies. To this aim, by both fluorescence-activated cell sorting (FACS) and fluorescence microscopy, we examined the occurrence of apoptotic markers such as caspase activity, Fas, p53 and Bcl-x in M540 bodies. In addition, we evaluated their ultrastructure by transmission electron microscopy. We found that M540 bodies express all the investigated markers, strongly supporting our hypothesis. We also found that M540 bodies contain fragmented DNA, another evidence of their apoptotic derivation. We investigated also the presence of M540 bodies in the different categories of patients. With respect to normozoospermic subjects, a higher content of M540 bodies was found in oligoasthenoteratozoospermic and asthenoteratozoospermic, but not in asthenozoospermic and teratozoospermic men. Interestingly, these subjects are those whose semen shows the highest levels of apoptotic signs. The variable occurrence of apoptotic bodies in semen may thus be considered a sign of abortive apoptosis in male reproductive organs. Of interest, since M540 bodies exhibit a similar size and density to sperm, they represent a confounding factor in FACS studies on ejaculated sperm.
Key words: apoptosis/human semen/M540 bodies
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Introduction |
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Human semen is a complex biological fluid in which heterogeneous cell types are present, including germ cells (other than mature spermatozoa) with different degrees of maturity and somatic cells. Recently we have reported the occurrence in semen of membrane surrounded round bodies, whose levels appear to be notably higher in subjects with decreased quality of semen (Muratori et al., 2004). We termed such bodies as M540 bodies, due to their bright staining with merocyanine 540 (M540). M540 is a lipophilic compound, sensitive to changes in the membrane architecture such those occurring during somatic apoptosis (Aussel et al., 1993; Mower et al., 1994) and capacitation of spermatozoa from some mammalian species (Gadella and Harrison 2000; Rathi et al., 2001), but not human (Muratori et al., 2004; Martin et al., 2005). Since M540 bodies are promptly stained with M540, their membrane is likely to be differently packaged from normal cell membranes. In addition, the failure of the nuclear stain YOPRO-1 (Y1) to label M540 bodies suggests their virtual lack of chromatin material (Muratori et al., 2004). Finally, M540 bodies appear to be heterogeneous elements in both size and density as indicated by light microscopy examination and by their distribution in different fractions of discontinuous density gradients (Muratori et al., 2004). Later, we showed that M540 bodies are ubiquitinated and that the amount of semen ubiquitination due to M540 bodies was sharply associated to poor seminal parameters (Muratori et al., 2005).
Most of the characteristics of M540 bodies prompted us to originally speculate that such bodies might be apoptotic bodies derived from testis apoptosis (Muratori et al., 2004). Morphologically, apoptosis is characterized by the condensation of nuclear chromatin into the periphery of nuclei, the shrinkage of the cell and, finally, the formation of membrane-encased bodies containing organelles (Kerr et al., 1994). Physiological apoptosis in testis involves all germ cell compartments (Oldereid et al., 2001) and is believed to play a pivotal role in tuning the balance between germ cells and Sertoli cells (Sakkas et al., 1999a). Increased germ cell apoptosis has been observed in many pathological conditions, such as during maturation arrest (Tesarik et al., 1998a; Jurisicova et al., 1999), after treatment with testicular toxicants (Tesarik et al., 2000; Boekelheide, 2005) or after hormone withdrawal (Tesarik et al. 1998b; Tesarik et al., 2002). The presence of apoptotic signs in ejaculated sperm has been reported in concomitance with testicular and systemic diseases (Baccetti et al., 1996; Gandini et al., 2000) and infertility or subfertility (Baccetti et al., 1997; Shen et al., 2002; Agarwal and Said, 2005). Apoptotic bodies, as detected by electron microscopy, have been shown in human ejaculates from subfertile men (Baccetti et al., 1996; Gandini et al., 2000). To explain the presence of apoptotic features in semen of subfertile patients, Sakkas et al. (1999a) proposed that testis apoptotic pathway may derail leading to a failure of the complete cell death programme.
The aim of the present study was to further characterize M540 bodies and investigate the hypothesis that they represent apoptotic bodies by examining the presence of apoptotic markers reported previously in ejaculated sperm from subertile patients (Sakkas et al., 2002), including caspase activity (Weng et al., 2002; Paasch et al., 2004), Fas (Sakkas et al., 1999b; McVicar et al., 2004; Stronati et al., 2006), p53 (Sakkas et al., 2002) and Bcl-x (Sakkas et al., 2002; Stronati et al., 2006) expression as well as the presence of DNA fragmentation. Finally, we evaluated their ultrastructure by transmission electron microscopy (TEM).
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Antibodies and other chemicals
The monoclonal antibodies (mouse immunoglobulins, Ig, G1) used in the present study were: ab-2, anti-Fas, used at dilution 1:40; Ab-11, anti-p53, used at dilution 1:50 (Calbiochem, Nottingham, UK) and H-5, anti-Bcl-xL, used at dilution 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Such antibodies were revealed by a goat anti-mouse IgG, conjugated with FITC (Sigma Chemical, St Louis, MO, USA). The rabbit polyclonal antibody against Bcl-xS/L (Santa Cruz Biotechnology), used at dilution 1:100, was revealed by a goat anti-rabbit IgG conjugated with FITC (SouthernBiotech, Birmingham, AL, USA). DNAse I was from Pharmacia Biotech Italia (Milan, Italy). Vybrant FAM Poly caspases Assay Kit was from Invitrogen (San Giuliano Milanese, Milan, Italy). Human tubal fluid (HTF) and modified HTF media were purchased from Celbio (Milan, Italy). Mouse IgG1 Isotype Control was from Exbio (Praha, Czech Republic). Normal Rabbit Serum was from DBA (Milan, Italy). Normal Goat Serum (NGS) and the other chemicals were from Sigma Chemical.
Semen sample collection and preparation
Semen samples were collected, according to World Health Organization criteria (World Health Organization, 1999), from subjects undergoing routine semen analysis for couple infertility in the Andrology laboratory of the University of Florence. The study was approved by Hospital Committee for Investigations in Humans, and informed consent was obtained by the patients. Since the aim of the present study was to characterize M540 bodies, we chose those samples in which such bodies occurred at high level, after checking by light microscopy in May–Grunwald stained samples.
For the experiments aimed to calculate the percentages of M540 bodies versus sperm in different categories of patients, 4 asthenozoospermic (A), 14 asthenoteratozoospermic (AT), 9 normozoospermic (N), 9 oligoasthenoteratozoospermic (OAT) and 18 teratozoospermic (T) samples were included. Experiments were performed in rough semen samples after washing twice with HTF medium. After washing (500 x g for 10 min), samples were fixed in paraformaldehyde [PF, 200 µl, 4% in phosphate-buffered saline (PBS) pH 7.4] for 30 min at room temperature, except for the Fas detection and caspase assay (see below). For Fas detection and caspase assay, samples were fixed after the immunofluorescence staining procedure and before the addition of propidium iodide (PI, see immunofluorescence section).
Determination of percentage of M540 bodies in subfertile patients
PF fixed semen samples were washed twice in HTF medium and double stained with M540 (13.5 µM) and Y1 (25 nM) in 500 µl of modified HTF containing 1 mg/ml of PVP (Muratori et al., 2004). Then samples were analysed by fluorescence-activated cell sorter (FACS). For each patient, two sperm suspensions were prepared for instrumental setting and data analysis: (i) by omitting both M540 and Y1 staining (nonspecific fluorescence sample) and (ii) by omitting only the M540 staining (sample for compensation, see below). Y1 green fluorescence and M540 red fluorescence were revealed by using, respectively, an FL-1 (515–555 nm wavelength band) and an FL-2 (563–607 nm wavelength band) detector of a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) equipped with a 15 mW argon-ion laser for excitation. Fluorescence compensation was set by acquiring samples labelled with only Y1. For each sample, 10 000 events were recorded in a FSC/SSC (Forward Scatter/Side Scatter) region excluding debris (R1, Fig. 1A). The number of M540 bodies was determined by counting the M540+/Y1– events in the R1 region. Sperm number was determined by counting the Y1+ events in the flame shaped region characteristic of sperm (Muratori et al., 2004). The amount of M540 bodies was expressed as percentage versus sperm number.
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To exclude the possibility that the fixation procedure modifies the percentage of M540 bodies, we also calculated the percentage of bodies before and after fixation. No difference was found between fresh and fixed samples (19.4 ± 1.9 versus 17.7 ± 3.4, mean ± SD, P = 0.22, n = 3).
Immunofluorescence for Fas, Bcl-xL/S, Bcl-xL and p53
For intracellular detection of p53, Bcl-xL and Bcl-xL/S, after washing twice with PBS/1%NGS, semen samples, adjusted to 5 x 106 spermatozoa, were incubated for 1 hour at 37°C with the primary antibodies in 100 µl of permeabilizing buffer (0.1% sodium citrate/0.1% Triton X 100). For detection of Fas, the same procedure was used but the buffer was PBS/1%NGS. After two further washes, samples were incubated with the FITC-labelled secondary antibodies (1:100, 1 h at room temperature) and washed again twice. Finally samples were resuspended in 400 µl of PBS and 8 µl of PI (0.6 µg/ml final concentration) were added. Fresh samples prepared for Fas detection were first fixed as described above and then labelled with PI.
Appropriate negative controls were obtained by omitting the primary antibody and by using mouse IgG1 isotype control (for Fas, p53 and Bcl-xL detection) or normal rabbit serum (for Bcl-xL/S detection). For each experiment set, a sample prepared with the immunofluorescence procedure but not labelled with PI was also prepared for fluorescence compensation in the FACS analysis (see below).
Caspase assay
Caspases were detected by Vybrant FAM Poly Caspases Assay Kit, which detects active caspases by using the FAM-VAD-FMK reagent, a fluorescently labelled inhibitor of such enzymes (FLICA). Briefly, fresh semen samples were resuspended at the concentration of 1 x 106 sperm/ml in 300 µl of HTF medium. Aliquots of 10 µl of 30x FLICA working solution were added and sperm suspensions were incubated for 1 h at 37°C and 5% CO2, in the dark. After two washes with Wash Buffer 1x (supplied by manufacturer), samples were fixed by adding 40 µl of 10% formaldehyde solution (supplied by manufacturer) for 10 min at room temperature. Hence, sperm samples were washed again twice and resuspended in 400 µl of Wash Buffer 1x containing 8 µl of PI for FACS analysis. For each test sample, two additional samples were prepared: (i) a negative control (absence of FLICA working solution) for data analysis; (ii) a test sample labelled only with FLICA (without PI staining) for fluorescence compensation in the FACS analysis (see below).
Terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling assay
DNA fragmentation was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick-end labelling (TUNEL) as described elsewhere (Muratori et al., 2003). Briefly, PF fixed semen samples adjusted to 5 x 106 spermatozoa were washed twice with 200 µl of PBS with 1% bovine serum albumin and permeabilized with 0.1% Triton X-100 in 100 µl of 0.1% sodium citrate for 2 min in ice. After washing two times, the labelling reaction was performed by incubating sperm in 50 µl of labelling solution (supplied with the In Situ Cell Death Detection Kit, fluorescein, Roche Molecular Biochemicals, Milan, Italy) containing the TdT enzyme for 1 h at 37°C in the dark. After labelling, two subsequent washes were performed and sperm were resuspended in 400 µl of PBS and 8 µl of PI (30 mg/ml in PBS) for FACS analysis. For each test sample, two additional samples were prepared: (i) a negative control (absence of TdT from the reaction mixture) for data analysis; (ii) a test sample labelled only by TUNEL assay (without PI staining) for fluorescence compensation in the FACS analysis (see below).
FACS analysis of DNA fragmentation, p53, Fas, Bcl-xL/S, Bcl-xL and caspase activity in M540 bodies
Green fluorescence of the investigated markers (DNA fragmentation, p53, Fas, Bcl-xL/S, Bcl-xL and caspase activity) was analysed in the population formed by solely M540 bodies. To distinguish bodies from nucleated cells, before FACS analysis, we treated samples with PI (see above) that labels cells but not M540 bodies (Muratori et al., 2005). Hence, we established in the FSC/SSC dot plots a region excluding debris (R1, Fig. 1A) and containing both M540 bodies and nucleated cells (i.e. sperm, large cells and cell aggregates). Then, within the R1 region, a second region (R2, Fig. 1B) containing the non-nucleated events was established in the PI fluorescence histogram. Analysis of green fluorescence was performed in R2 region, thus containing only M540 bodies. FITC green fluorescence and PI red fluorescence were revealed by using, respectively, FL-1 and FL-2 detectors of the FACScan flow cytometer. Fluorescence compensation was set by acquiring samples labelled only with green fluorescence but not with the red one (i.e. PI fluorescence). For each sample, 8000 events in the R2 region (see above) were recorded. Data were processed with Lysys II software (Becton Dickinson). For each investigated marker, the percentage of labelled M540 bodies in the test sample was determined by using the fluorescence histogram of the corresponding negative sample: (i) sample incubated with isotype control for Fas, p53 and Bcl-xL detection; (ii) sample incubated with normal rabbit serum for Bcl-xS/L detection; (iii) sample incubated in absence of the enzyme TdT for DNA fragmentation detection and (iv) sample incubated in absence of FLICA for caspase assay. The percentage of labelled M540 bodies was determined by setting a marker including >99% of the events in the frequency histogram of the negative control. Hence, all the events out of this region were considered positive.
In experiments aimed to compare (i) DNA fragmentation before and after nuclease treatment; (ii) expression of Bcl-xL/S and Bcl-xL in the same semen samples, results were expressed as mean values of fluorescence distribution.
Fluorescence microscopy
For observation by fluorescence microscopy, double-stained sperm were smeared on slides and examined using a fluorescence microscope (type 307-148002; Leitz, Wetzlar, Germany) equipped with E4 and N2.1 filters (Leica, Milan, Italy) by an oil immersion x100 magnification objective. Images were captured by a Canon digital camera using Remote Capture software (provided by Canon, Japan) and edited by Adobe photoshop version 5.0 (Adobe Systems Inc., CA, USA).
Transmission electron microscopy
After two washes with PBS 1x, semen samples were fixed for 1 h at room temperature in Karnovsky fixative [2.5% glutaraldehyde, 0.1% PF in 0.1 mol/l cacodylate buffer (pH 7.4)]. Then samples were postfixed with 1% buffered OsO4 in the same buffer, dehydrated, and pellets were embedded in Epon 812 (Fluka Chemical Co., Buchs, Switzerland). Ultrathin sections were stained by uranyl acetate followed by lead citrate. Observation was performed with a Phillips 410 electron microscope.
Statistical analysis
Data were analysed with Microcal Origin software, 6.1 version (MicroCal Software Inc., Northampton, MA, USA). Results are shown as the mean ± SD. Analysis of variance and the Student’s t-test were used to assess statistically significant differences: (i) between DNA fragmentation in M540 bodies, before and after treatment with nuclease (paired data); (ii) between the percentages of M540 bodies in the different categories of patients (independent data).
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M540 bodies in semen from different categories of patients
In our previous paper, we reported the percentages of M540 bodies (with respect to the total population of M540 bodies + spermatozoa) in semen from subfertile subjects (Muratori et al., 2004). In that study, the percentage of M540 bodies in the semen samples was evaluated in the flame-shaped region of FSC/SSC dot plot characteristic of spermatozoa. Here, we re-evaluated the percentage of M540 bodies in semen samples from A, AT, N, OAT and T subjects, considering the entire population of M540 bodies in semen and referring it to the population formed by solely sperm. Indeed, M540 bodies show dimensions ranging from those similar to sperm head to much larger, as demonstrated by both FACS and fluorescence microscopy (see further sections). Figure 2A shows the population of M540 bodies (M540+/Y1– events) as determined by double staining with M540 and the nuclear probe, Y1. In Fig. 2B, the morphometric characteristics (i.e. the FSC and SSC signals) of such population are shown. Note that M540 bodies are present both within and out of the flame-shaped region characteristic of spermatozoa (R2 in Fig. 2B). To check whether the centrifugation steps affected the composition of M540 bodies in the test sample, we collected all the supernatants and stained them with M540/Y1 together with the corresponding pellets. Then, we determined M540 bodies in the R2 region (smaller bodies) and in the R1 region (total bodies) both in the supernatants and pellet fractions. Although both bodies and spermatozoa are found in the collected supernatants, no difference was detected in the percentages of M540 bodies (versus total semen components) included in R1 and R2 regions, between supernatants and corresponding pellets (not shown). Hence, we conclude that the procedures do not affect the composition of M540 bodies in the test samples.
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In Fig. 2C, percentages of M540 bodies versus spermatozoa in normal (N) subjects and subfertile patients are shown. M540 bodies are present in all categories of subjects. The highest levels of M540 bodies are found in AT (P < 0.01 versus N) and OAT subjects (P < 0.05 versus N). In the latter, the amount of M540 bodies occasionally reaches dramatic values (up to 400%).
Immunofluorescence for Fas, p53, Bcl-xL/S and Bcl-xL.
Detection of Fas receptor, p53 and Bcl-xL/S in M540 bodies was performed by immunofluorescence coupled to FACS in PI labelled samples, after the exclusion of nucleated cells (PI positive events) and debris. The expression of Fas receptor, p53 and Bcl-xL/S in M540 bodies was confirmed also by fluorescence microscopy. Results indicate that M540 bodies express the three markers in all the samples investigated. Figures 3, 4 and 5 show representative frequency histograms (A) and the microscopic images (B, upper panels) of green fluorescence respectively for Fas receptor, p53 and Bcl-xL/S in M540 bodies. The latter are easily recognizable by lack of, or poor, PI staining (B, middle panels) and round shape (B, bright fields in the lower panels). Negative control is reported in C of the three figures. In Fig. 3B, two spermatozoa are present, one negative for Fas fluorescence and the other labelled in the post-acrosomal region, as reported by other authors (McVicar et al., 2004). In Fig. 4B, two spermatozoa negative for p53 expression are also present, whereas of the four spermatozoa present in Fig. 5B, three show Bclx labelling in the post-acrosomal region. Table 1 reports the mean (±SD) percentages of positive M540 bodies for Fas receptor, p53 and Bcl-xL/S, as determined by FACS in several experiments. Since immunofluorescence for Bcl-x reveals both the long (xL) and the short form (xS), we performed FACS analysis of the long form (Bcl-xL) in the same samples processed for Bcl-xL/S detection. Results indicate that the mean values of fluorescence due to Bcl-xL in M540 bodies are lower than those due to Bcl-xL/S (respectively: 4.0 ± 3 versus 7.0 ± 3.6, n = 5), indicating that M540 bodies express both forms of Bcl-x including the pro-apoptotic one, Bcl-xS.
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Caspases activity
To detect caspase activity in M540 bodies, we double stained semen samples with the generic probe FLICA and PI and evaluated them by FACS, after the exclusion of nucleated cells (PI positive events) and debris. In Fig. 6A, a representative fluorescence histogram of caspases detection is reported. Caspase activity in M540 bodies was observed also by fluorescence microscopy. Figure 6B shows the images of green fluorescence due to FLICA in M540 bodies. In the figure, fluorescence due to caspase activity is shown also in the mid-piece region and tail of sperm, as reported by other authors (Marchetti et al., 2004). Again, the corresponding red PI fluorescence (Fig. 6B, middle panels) and bright fields (Fig. 6B, lower panels) are reported. Images from negative controls (absence of FLICA) are reported in Fig. 6C. In Table 1, the percentages (mean ± SD) of M540 bodies exhibiting caspase activity are reported.
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TUNEL assay
All the investigated apoptotic markers resulted expressed in M540 bodies, indicating that such bodies are apoptotic bodies. To further investigate this hypothesis, we tested whether M540 bodies exhibit DNA fragmentation, despite of lack of staining with nuclear probes (Muratori et al., 2004, 2005 and present study). DNA fragmentation in M540 bodies was investigated by TUNEL assay coupled to FACS in samples stained with PI, after the exclusion of nucleated cells (PI positive events) and debris. As shown in Fig. 7A, which reports a representative fluorescence histogram of TUNEL assay, a fraction of M540 bodies contained fragmented DNA. To further ascertain that the fluorescence signal due to TUNEL labelling in M540 bodies was really due to the occurrence of DNA fragmentation, we labelled some samples by TUNEL assay and perform FACS analysis before and after the treatment with DNAse I. Results indicate that the mean values of fluorescence distribution increase after nuclease treatment (12.0 ± 10.0 versus 22.2 ± 18.4, P < 0.05, n = 6). We conclude that a variable fraction of M540 bodies contains fragmented DNA (see Table 1). DNA fragmentation in M540 bodies was observed also by fluorescence microscopy. Fig. 7B shows the images of green fluorescence due to TUNEL labelling in M540 bodies. Again, the corresponding red PI fluorescence (Fig. 7, middle panels) and bright fields (Fig. 7, lower panels) are reported. Images from negative controls are shown in Fig. 7C.
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Evaluation by TEM
We evaluated the ultrastructure of semen components by TEM in ejaculates of subjects containing high level of M540 bodies. Figure 8 shows two round bodies found in two different subjects. As shown, they closely resemble apoptotic bodies previously found in human semen from subefertile subjects (Baccetti et al., 1996; Gandini et al., 2000). Whether these elements are M540 bodies cannot be unequivocally deduced, however their dimensions, similar to sperm heads (Fig. 8), suggest this possibility.
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Discussion |
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In this study, we report consistent evidence that M540 bodies, round structures present in semen (Muratori et al., 2004), represent apoptotic bodies. Indeed, we clearly show that M540 bodies express several markers, such as Fas receptor, p53, Bcl-x, caspase activity and fragmented DNA, known to have a role in testis and somatic apoptosis. In addition, we report that the levels of such bodies are much higher in semen of OAT and AT men, where the highest levels of semen apoptotic signs have been found (Sakkas et al., 1999b). Further, ultrastructural features of round elements from semen samples of subjects containing high levels of M540 bodies are consistent with those described for apoptotic bodies. We believe that the demonstration of occurrence of high levels of apoptotic bodies in semen has important clinical and methodological implications. Indeed, their presence in semen may be considered a sign of pathological/excessive apoptosis in the testis or, in general, in the male genital tract, and at the same time, raises methodological issues in studies performed by FACS.
The molecular mechanisms responsible for testis apoptosis have not yet been completely elucidated, although they do not appear to substantially differ from apoptosis in other tissues. Pro-apoptotic stimuli in the testis are variable and can occur both in physiological and pathological conditions (Koji and Hishikawa, 2003). The different testis pro-apoptotic stimuli may induce different pathways (including the Fas-mediated and the mithochondrial-dependent pathways) to trigger the programme of cell death (Hikim et al., 2003). All the stimulated apoptotic pathways converge at the level of caspases, which are considered the key enzymes of apoptosis, acting both as initiators and executors of the programme of cell death. Similarly, the breakage of the double stranded DNA is an ubiquitous step in the sequence of apoptosis. Other proteins involved in the cell death programme have been demonstrated during testis apoptosis: Fas (Lee et al., 1997, 1999), members of the Bcl-2 family (Joshi and Dighe, 2006) and p53 (Boekelheide, 2005). We show here that M450 bodies contain all these markers to variable extents. In particular, caspase activity is detected in a very large percentage of M540 bodies, whereas the other apoptotic markers were present only in a fraction of them. This finding is not surprising, since activation of caspases is the final step of the process in which all the different apoptotic signalling pathways converge. In addition, the FLICA used to detect activated caspases in our study reveals most of the known caspases.
At the beginning, the finding that M540 bodies exhibit DNA fragmentation was quite surprising because, in our original study, M540 bodies were detected and distinguished from sperm in FACS, due to their lack of staining with nuclear probes (Muratori et al., 2004, 2005 and present study). However, in apoptotic bodies DNA may be absent or present in low amount (due to nuclei segmentation) with intensive fragmentation (due to nuclease activation). Since the intensity of PI staining is largely dependent on DNA content, whereas that of TUNEL labelling depends on the numbers of DNA breaks, it is not surprising that apoptotic bodies are poorly stained with PI and brilliantly stained with TUNEL (see e.g. Fig. 7, in which some PI staining is present in one of the shown bodies). In addition, when DNA is intensively fragmented (it occurs following apoptosis), the avidity for intercalating probes (which is elevated for coiled DNA due to the fact that binding with them reduces free energy of torsion stress) is almost completely lost.
The presence of apoptotic bodies has been already reported in human ejaculates, in TEM investigations of morphological characteristics of human spermatozoa (Baccetti et al., 1996; Gandini et al., 2000). In addition, occurrence of apoptotic signs in ejaculated sperm has been extensively reported in recent years. Such signs are mainly detected in subfertile patients and include caspase activity (Weng et al., 2002; Paasch et al., 2004), expression of key apoptotic proteins (Sakkas et al., 2002) and apoptosis-like ultrastructures (Baccetti et al., 1996). Even sperm DNA fragmentation is considered to be a sign of testicular apoptosis, although other events can explain its occurrence (Muratori et al., 2006). An impairment in the process of testicular apoptosis has been hypothesized and termed ‘abortive apoptosis’ by Sakkas et al. (1999a) to explain the presence of such apoptotic features in semen. Consistent with this theory, M540 bodies could represent apoptotic bodies derived from abortive testis apoptosis. However, apoptotic bodies may derive also from other cell types undergoing apoptosis in the male genital tract, such as epithelial cells of the epididymis or of the ejaculatory ducts. In this light, the high levels of apoptotic bodies found in semen of OAT and AT subjects might indicate that deregulated/excessive apoptosis of the male genital compartments, particularly of the testis, is characteristic of these subjects. However, further investigation is needed to understand whether apoptosis may be responsible for, or a consequence of, the low semen quality. Further investigation is also necessary to understand why M540 bodies (and ejaculated sperm with signs of apoptosis) are not eliminated as usually occurs in the apoptotic programme.
Occurrence of M540 bodies in human semen at variable (and sometimes high) extents and the finding that they are apoptotic residues raises methodological issues in studies on ejaculated sperm conducted by FACS. Indeed, since a fraction of M540 bodies are very similar in size and density to sperm (Muratori et al., 2004), they cannot be directly separated from sperm by using FSC/SSC signals, generally used to gate the population of interest. Hence, their presence may be somehow misleading when analysing sperm populations by FACS as we have recently shown (Muratori et al., 2005). In this light, FACS studies aimed to investigate apoptotic as well as other markers in ejaculated sperm should carefully consider that M540 bodies might also be labelled and included in the population of interest.
In conclusion, the round membrane-surrounded bodies observed in semen are likely to be apoptotic bodies. Although we are not able to unequivocally demonstrate at present that they originate from the testis, it is likely that they may derive from a process of testicular abortive apoptosis, as speculated for ejaculated sperm with apoptotic signs (Sakkas et al., 1999a). The variable occurrence of cell (apoptotic) residues may thus be considered a sign of deregulated apoptosis in the male reproductive organs. In addition, their presence in semen may represent a bias in FACS studies of ejaculated sperm.
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Acknowledgements |
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We are indebted to Prof. Paola Piomboni (University of Siena, Italy) for expert advice. We also thank Drs Erminio Filimberti and Selene Degl’Innocenti for help in selection of samples and Dr Pasquale Imperiale for technical advice. This work was supported by a grant from the Ministry of University and Scientific Research (PRIN project to E.B.).
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Submitted on May 11, 2007; accepted on May 22, 2007.
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