Division of Endocrinology, Andrology and Internal Medicine, Department of Biomedical Sciences, University of Catania, Catania, Italy
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Abstract |
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Key words: aneuploidy/chromatin structure/diploidy/globozoospermia/sperm
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Introduction |
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Two types of globozoospermia have been described (Singh, 1992). Type I is characterized by a complete lack of acrosome and acrosomal enzymes and by a spherical arrangement of the chromatin. These sperm are unable to penetrate the zona pellucida, causing primary infertility. Type II has some acrosomal covering with a conical nucleus, which may be surrounded by large cytoplasmic droplets indicating secondary degenerative changes. It is assumed that infertility in this type of globozoospermia is caused by subsequent poor motility (Singh, 1992
).
Sperm DNA status in those patients with globozoospermia is not well known. Carrell and collaborators have found an elevated sperm aneuploidy rate, studied by fluorescence in-situ hybridization (FISH), for chromosomes 13, 21, X and Y (Carrell et al., 1999) and 15 and 18 (Carrell et al., 2001
) in one of two infertile siblings with globozoospermia, whereas a normal aneuploidy rate has been reported by other authors (Viville et al., 2000
). A recent case report has shown that only 13% of sperm demonstrated DNA denaturation following the application of sperm chromatin structure assay (SCSA). A similar percentage has been obtained by using the COMET assay (Larson et al., 2001
) These results were similar to those obtained in normal controls, suggesting that globozoospermia is not associated with abnormal chromatin structure or DNA strand breaks. However, globozoospermia is a severe form of teratozoospermia and as such it is possible that round-headed sperm may have an abnormal chromatin structure and/or DNA strand breaks both of which have been reported to cause early pregnancy loss (Ibrahim et al., 1988
) or to increase the risk of malformations, cancer and genetic diseases in the offspring (Potts et al., 1999
). Therefore, the evaluation of the sperm DNA status in patients with globozoospermia is fundamental.
This study reports the sperm DNA status of a patient with globozoospermia. The sperm chromatin packaging quality was assessed by applying propidium iodide (PI) staining, whereas DNA fragmentation was studied by using the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling (TUNEL) procedure. The sperm aneuploidy rate for chromosome 8, 12, 18, X and Y was also evaluated by means of FISH.
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Patients and methods |
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TUNEL assay
Sperm were labelled using the Apoptosis Mebstain kit (Beckman Coulter), purchased from Instrumentation Laboratory (Milan, Italy). To obtain a negative control, TdT was omitted from the reaction mixture. The positive control was obtained by pre-treating the sperm with 1 µg/ml deoxyribonuclease I, RNAse-free (Sigma Chemical) at 37°C for 60 min before labelling. DNA fragmentation was evaluated by FCM. Ten thousand events were measured for each sample at a flow rate of 500 events/s. The debris was eliminated following the same procedure. Light-scattering and fluorescence data were obtained at a fixed gain setting in logarithmic mode. The percentage of FITC-labelled sperm was determined in the FL-1 channel of the FCM.
FISH analysis
Sperm FISH analysis was performed as previously reported (Calogero et al., 2001). Briefly, sperm were washed three times in phosphate buffered saline (PBS), pH 7.2, centrifuged at 650xg for 10 min and fixed in methanol/acetic acid (3:1). They were spread on slides and the slides were washed in 2x standard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25 mmol/l dithiothreitol.
Double- and a triple-colour FISH were carried out, using -centromeric probes for chromosomes 8, 12, 18, X and Y, purchased from the University of Bari (Bari, Italy). The probe mixture for triple FISH consisted of a repetitive DNA sequence of centromeric probes: chromosome X (pDMX1), labelled with FITC; for chromosome Y (pLAY5.5), labelled with Cy3; and for chromosome 12 (pBR12), labelled with FITC and Cy3. The probe mixture for double-colour FISH also consisted of a repetitive DNA sequence of centromeric probes for chromosome 8 (pZ8.4) and for chromosome 18 (2Xba), labelled FITC and Cy3, respectively.
The slides were denatured in a PCR machine with a solution of 70% formamide/2xSSC (pH 7.5) at 80°C for 150 s, immersed in a 70, 90 and 100% ethanol series for 3 min each and dried by air. The probes, precipitated and denatured at 80°C for 8 min, were applied directly to the slides which were then covered with a coverslip and sealed with rubber cement. Hybridization occurred overnight in a dark humidified container at 37°C, after which one coverslip was removed and the slides were immersed in a post-hybridization wash of 50% formamide/2xSSC for three times at 37°C for 5 min, 2xSSC for three times at 42°C for 5 min and 2xSSC/0.1% Tween 20 at room temperature for 5 min. The slides were then mounted in 4',6-diamidino-2-phenylindole (DAPI) counterstain and antifade. The slides were observed using an Axiophot fluorescent microscope (C.Zeiss, Oberkochen, Germany) with an appropriate set of filters, namely single band DAPI, FITC and Cy3. Only intact sperm bearing a similar degree of decondensation and clear hybridization signals were scored; disrupted or overlapping sperm were excluded from analysis. Sperm were regarded as abnormal if they presented two (or more) distinct hybridization signals for the same chromosome, each equal in intensity and size to the single signal found in normal monosomic nuclei. We considered only clear hybridization signals, similar in size, separated from each other by at least one signal domain and clearly positioned within the sperm head. Divided (split) signals were not scored as disomies. Sperm were scored as nullisomic if they showed no signal for a given chromosome, when the signal of the other chromosome tested was present. Finally, a spermatozoon was considered diploid in cases where it manifested two signals for each tested chromosome and when the tail, as well as the normal oval shape of a sperm head, were evident. The absence of FISH signals in a spermatozoon head showing DAPI stain was considered to be a case of no hybridization.
Statistical analysis
Data are shown as mean ± SEM. Statistical analysis was carried out using unpaired Student's t-test. P-value < 0.05 was considered to be significant.
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Results |
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PI staining
Two different cell populations were detected in the semen sample of the patient with globozoospermia and in normal controls (Figure 1). The first fluorescence peak corresponded to sperm with mature chromatin, whereas the second peak corresponded to haploid cells with an increased DNA accessibility to PI (sperm with immature chromatin). A third peak including diploid cells was also observed. The percentage of sperm with immature chromatin was significantly higher (P < 0.001) in the patient with globozoospermia (Table II
).
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Discussion |
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Our patient with globozoospermia had an elevated number of sperm with decondensed chromatin. The assessment of DNA fragmentation showed a percentage of TUNEL-positive sperm greater than that found in normal controls, suggesting an increased rate of apoptosis. However, it is noteworthy that cells showing a high degree of DNA fragmentation are not necessarily apoptotic (Evenson et al., 2002). These results indicated that, like other forms of teratozoospermia (Gandini et al., 2000
), globozoospermia is probably associated with abnormal chromatin structures and DNA strand breaks. An increased rate of DNA fragmentation has also been reported by Baccetti and co-workers in patients with round-headed sperm (Baccetti et al., 1996
). Our data are, however, at variance with a recent case report showing a normal chromatin packaging in a patient with globozoospermia (Larson et al., 2001
). Although we do not have a definitive explanation for this, it may relate to the different methods employed to evaluate chromatin integrity (SCSA and single cell gel electrophoresis assay versus TUNEL). Alternatively, it may relate to the fact that only 69% of the sperm were found to have round heads in the patient reported in the latter study whereas all the sperm in our patient were globozoospermic. In addition, it should be highlighted that our patient, unlike the patient in a previous study (Larson et al., 2001
), also had asthenozoospermia and a direct relationship between poor motility and the degree of DNA fragmentation has recently been suggested (Muratori et al., 2000
).
Syms and co-workers (1984) have demonstrated that globozoospermic sperm could undergo nuclear decondensation when incubated with crushed hamster oocytes (Syms et al., 1984). Decondensation and pronuclear formation have also been detected after direct injection into hamster oocytes (Lanzendorf et al., 1988
). Thus, if the inability to penetrate the oocytes is bypassed, fertilization could take place. This was made possible in humans with the introduction of the intracytoplasmic sperm injection (ICSI) technique (Palermo et al., 1992
). Following the first pregnancies obtained from patients with globozoospermia by means of ICSI (Liu et al., 1995
; Trokoudes et al., 1995
), a number of globozoospermic patients have undergone this type of treatment. However, a low fertilization rate has been reported, unless oocytes have been activated with calcium chloride or ionophore (Battaglia et al., 1997
; Rybouchkin et al., 1997
; Stone et al., 2000
) which implies a defective capability of the globozoospermic spermatozoon to activate the oocyte. We hypothesise that the poor status of chromatin condensation and DNA fragmentation may also contribute to the low fertilization rate reported in these patients. Accordingly, a poor chromatin packaging and/or damaged DNA have been implicated in the failure of sperm decondensation after ICSI, resulting in fertilization failure (Sakkas et al., 1996
). Moreover, its has been shown that patients with male factor infertility display higher levels of loosely packaged DNA (Evenson et al., 1980
; Foresta et al., 1992
; Sailer et al., 1995
; Golan et al., 1997
).
Sperm aneuploidy is another abnormality which may impair ICSI outcome (Calogero et al., 2001). The normal sperm aneuploidy rate found in the globozoospermic patient reported in this study ruled out this hypothesis in this case. Accordingly, other studies have reported that the incidence of chromosomal abnormalities in round-headed sperm is similar to that in sperm from a fertile donor (Kullander and Rausing, 1975
; Lalonde et al., 1988
; Viville et al., 2000
). However, a contribution of sperm aneuploidy to the low ICSI outcome success rate cannot be completely excluded in patients with globozoospermia since an elevated sperm aneuploidy rate has also been reported in one of two infertile siblings with globozoospermia indicating multigenic defects and/or variable expression of the syndrome (Carrell et al., 1999
, 2001
). Recently, these authors have reported the first case of sperm chromosome-15 aneuploidy in a patient with variable familial expression of round-headed morphology who had a child with trisomia 15 following ICSI (Carrell et al., 2001
).
In conclusion, we have found an association between globozoospermia and abnormal sperm chromatin packaging and DNA strand breaks. The reported lower success rate in ICSI programmes for patients with globozoospermia (Battaglia et al., 1997; Rybouchkin et al., 1997
; Stone et al., 2000
) may be explained by this finding and this should be kept in mind when patients with this type of teratozoospermia are enrolled in ICSI programmes.
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Acknowledgements |
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Notes |
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References |
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Submitted on February 2, 2002; resubmitted on March 19, 2002; accepted on May 10, 2002.