1 Laboratoire de Biogénotoxicologie et Mutagenèse Environnementale (EA1784), IFR PMSE112, Faculté de Médecine Timone, 27, Boulevard Jean Moulin, 13385, Marseille cedex 05, 2 Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5 and 3 Laboratoire d'enseignement et de recherche sur le traitement de l'Information Médicale (LERTIM), Faculté de Médecine, 27, Boulevard Jean Moulin, 13385 Marseille cedex 05, France
4 To whom correspondence should be addressed: Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5, France. Email: mguichaoua{at}ap-hm.fr
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Abstract |
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Key words: asynapsis/meiosis/pachytene checkpoint/pachytene stage/spermatogenic failure
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Introduction |
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Materials and methods |
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Immunofluorescence
Meiotic cells from nine of the above patients (five in the NO group and four in the O group) and one control were immunostained according to the technique described by Metzler-Guillemain and Guichaoua (2000). Two antibodies were applied: (i) a rabbit anti-hamster synaptonemal complex polyclonal antibody which recognizes the Cor1 protein of the axial core/lateral element of the synaptonemal complex (SC) at a dilution of 1/1000 (kindly provided by Peter Moens and Barbara Spyropoulos); (ii) an anti-kinetochore serum from a patient having a CREST syndrome was used at dilution of 1/20 (kindly provided by Marielle Sanmarco). Two secondary antibodies (Sigma) were applied: a fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG at a dilution of 1/200 in phosphate-buffered saline (PBS) and a tetramethylrhodamine B isothiocyanate (TRITC)-conjugated goat anti-human IgG at a dilution of 1/16 in PBS for 1 h at 37°C.
Microscope analysis
After Giemsa staining, pachytene nuclei were analysed using a Zeiss Axioplan 2 photomicroscope (Zeiss, Germany) with a x 100 Plan apo objective. Analysis of immunofluorescent preparations was performed using the same microscope equipped with an epifluorescent system. Selected nuclei were then examined with a confocal scanner Leica TCS 4D mounted on a Leica DMIRBE microscope (Leica, Germany).
The number of pachytene spermatocytes analysed per patient varied from 20 to 100, with the exception of patient TeH 21 (17 nuclei analysed) (Table II) depending on the richness of the individual preparations. The pachytene nuclei were selected and analysed on the basis of good spreading of the bivalents and sex chromosome configuration, allowing unambiguous evaluation of pairing failure. For each patient, we quantified the number of pachytene nuclei showing asynapsis, which is defined as the pairing defect of either homologous chromosomes or segments of homologous chromosomes. These nuclei were termed asynapsed nuclei. Immunostained pachytene nuclei were classified as early or late substages, according to the different morphologies of the XY pair, based on the classification of Solari (1980).
Statistical analysis
For each patient, we evaluated an index of asynapsis which corresponded to the ratio: number of nuclei with asynapsed bivalents/number of nuclei analysed. We then used the Mann and Whitney U-test. We compared the results of the meiotic studies from the obstructive (O) group with the non obstructive (NO) group, and with controls. Statistical analysis was performed using SPSS 11.1 software.
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Results |
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Severe alterations of spermatogenesis were observed in all patients of the NO group (Table II). All men produced very few or no sperm in the testis. Four patients (TeH 9, TeH 17, TeH 21 and TeH 46) had Sertoli cells only at the histological level. In all the other patients, numerous Sertoli cell only tubules were seen on the preparations; the other tubules showed hypospermatogenesis with few sperm. Four patients (TeH 2, TeH 16, TeH 52, TeH 66) were mosaic at the histological level, also showing tubules with normal spermatogenesis. Two patients (TeH 3 and TeH 10) had a maturation arrest, showing interrupted spermatogenesis at round spermatid stage. No sperm were seen in these two patients at the histological level, but both had sperm in the ejaculate. Impaired fibrous and/or oedematous interstitial tissue, and hyalinose of the tubule walls were reported in the majority of the patients.
Meiotic study
The results are summarized in Table III. A total of 2931 pachytene nuclei were analysed, 1354 for the O group and 1577 for the NO group. The mean number of pachytene nuclei analysed per patient was 58 for both groups. The mean rates of asynapsed nuclei in the O group and the NO group were 9.8 and 25.4% respectively, and the difference between the two groups was statistically significant (P<0.001). In total, 855 nuclei were analysed for the 14 controls, with a mean of 59 nuclei analysed per control. The mean rate of asynapsed nuclei in this group was 10.6%.
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Immunofluorescence
Nine patients and a control were analysed using this staining, and the characteristics of the asynapsis were as observed with Giemsa staining (Figure 1C and D). The mean number of pachytene nuclei analysed per patient was 70 for the O group and 63 for the NO group. In the O group, the percentage of asynapsed nuclei varied from 2.4 to 9.5%, with a mean of 6%; in the NO group, the percentage of asynapsed nuclei varied from 7.6 to 26.9%, with a mean of 17.2% (Tables III and IV). The difference between the two groups was statistically significant (P=0.032). The percentage of asynapsis in the control for this protocol was 11.1%.
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Discussion |
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It was suggested by de Boer et al. (2004) that zygotene may often be misinterpreted as asynapsed pachytene, while de Vries et al. (1999)
refer to the possibility of an extended zygotene stage. Sex vesicle analysis, however, does allow an exact identification of the pachytene stage, and applying either interpretation, accumulation of zygotene or early pachytene spermatocytes is an indication that progression of meiosis is defective. It is likely that in most cases this is a secondary effect, and this is clearly shown by our observation that early stages are more common than late in the members of the two groups tested by immunocytochemistry. Indeed, in this study, staging of pachytene cells, based on the morphology of the XY chromosome pair, showed a prevalence of early pachytene substages in the O group (83.5%) and in the NO group (78.1%), whereas in the control group, late pachytene nuclei were in the majority (53.9%), as has previously been reported by Solari (1980)
and de Boer et al. (2004)
. Thus, although the asynapsis rates were the same in both the O group and in controls, the finding that early pachytene substages consistently prevail in both the O group and the NO group suggests that meiotic germ cell development is impaired even in cases of obstructive infertility. Moreover, the prevalence of early pachytene nuclei in patients of the two groups suggests that the pachytene checkpoint is localized in mid-pachytene in humans and acts effectively in patients with meiotic anomalies. However, the fact that sperm were present at the time of meiotic study in patient TeH 3 suggests that the pachytene checkpoint is not an absolute barrier. Indeed, with the exception of Aran et al. (2003)
, the majority of studies show that abnormal meiotic cells can complete spermatogenesis. A significant increase of aneuploidy, disomy and diploidy in sperm was shown by sperm FISH analysis for infertile men (Moosani et al., 1995
; Bernardini et al., 1998
; Pang et al., 1999
; Calogero et al., 2001
; Schmid et al., 2003
) and for a men carrying a mutant allele of the mismatch repair gene, hMSH2 (Martin et al., 2000
). In the same way, fertilization and pregnancy rates in obstructive azoospermia were higher than those achieved in non-obstructive azoospermia (Palermo et al., 1999
; Balaban et al., 2001
; Vernaeve et al., 2003
). Thus, qualitative and quantitative analyses of meiotic abnormalities are essential for the identification of meiotic abnormalities which could be directly responsible for the spermatogenic failure and thus reveal new aetiologies of male infertility. The panel of antibodies which detects individual protein components at different stages of meiosis provides a valuable tool for the detection and interpretation of abnormal meiotic profiles (Sun et al., 2004
).
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Acknowledgements |
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References |
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Submitted on March 15, 2004; resubmitted on February 22, 2005; accepted on February 24, 2005.
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