Hum. Reprod. Advance Access originally published online on April 11, 2007
Human Reproduction 2007 22(6):1567-1572; doi:10.1093/humrep/dem045
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Meiotic abnormalities in patients bearing complete AZFc deletion of Y chromosome
1 Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147 Bd Baille, 13385 Marseille cedex 05, France 2 Laboratoire Biologie de la Reproduction, Hôpital Nord, 42055 Saint Etienne, France 3 Laboratoire de Biogénotoxicologie et Mutagenèse Environnementale (EA 1784), IFR PMSE 112, Faculté de Médecine Timone, 27 Bd Jean Moulin, 13385 Marseille cedex 05, France
4 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, Hôpital de la Conception, 147 Bd Baille, 13385 Marseille cedex 05, France. E-mail: mguichaoua{at}ap-hm.fr
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Abstract |
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BACKGROUND: We studied meiosis in three infertile patients presenting complete AZFc microdeletion and three controls.
METHODS: Primary spermatocytes were immunolabeled with SCP3, BRCA1 and 
H2AX. We quantified the leptotene, zygotene and pachytene stages, and pachytene abnormalities: asynapsis and fragmented and dotted synaptonemal complexes (SCs).
RESULTS: SCP3 level was significantly higher in leptotene and zygotene (bouquet) stages in patients, suggesting AZFc may have a direct effect on early prophase. SCs were abnormal in 77.3% of pachytene nuclei of patients versus 30.8% of controls. The two groups differed significantly (P < 0.001) in asynapsed nuclei, fragmented SC and dotted SCs. In patients, asynapsis were short and limited to a few bivalents. Staging of pachytene nuclei based on the morphology of the XY pair with BRCA1 revealed a prevalence of early pachytene substages (70.7%) in patients. H2AX was normally phosphorylated.
CONCLUSIONS: In the absence of the AZFc region, the transient zygotene stage is extended, and chromosome condensation is reduced. The low level of limited asynapsis, the normal H2AX staining and the incomplete loss of germ cells at the pachytene checkpoint indicate that the AZFc region is not critical for meiotic recombination. We suggest that the pachytene phenotype develops secondarily to a primary defect that influences meiosis.
Key words: asynapsis/AZFc deletion/meiosis/pachytene checkpoint/SC fragmentation
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Introduction |
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Y chromosome microdeletions are one of the most common causes of male infertility, and AZFc microdeletion is the most frequent (Krausz et al., 2003
). Deletion of AZFc is associated with either severe oligozoospermia, or azoospermia, but it is unknown how this specific microdeletion leads to spermatogenic failure. The AZFc region spans 3.5 Mb and contain 21 genes, including four deleted in azoospermia (DAZ) genes that are implicated in the regulation of mRNA translation (Lin et al., 2006). However, it is admitted that the structure around AZFc is highly polymorphic and that partial deletions can also be associated with impaired spermatogenesis (Repping et al., 2003). Moreover, one should also consider exposure to environmental factors and associated causes of infertility. So, it is now necessary to clarify the genotype–phenotype correlations and to describe the testicular phenotype that corresponds to AZFc microdeletions. Several approaches have been used to describe spermatogenic defects. Spermiograms show that deletion of the AZFc interval is associated with either azoospermia, or severe to mild oligospermia and insufficient production of mature sperm to enable reproduction (Vogt, 2005). When testicular biopsy material is available, histopathology reflects a spectrum of spermatogenic phenotypes from Sertoli-Cell-Only Syndrome to hypospermatogenesis with or without meiotic or maturation arrest. Yet, these two methods of meiotic investigation do not show up the specific causes of spermatogenic failure. Direct meiotic study using multicolor Fluorescence in-situ hybridization (FISH) for both centromere and telomere regions has been performed to evaluate whether the AZF-deleted segment influences the ability of the Y chromosome to pair (Yogev et al., 2004). In the present study, we also focused on meiosis, but with a different approach because we hoped that insight into meiotic chromosome behavior may provide greater understanding of the mechanisms of spermatogenic failure in the AZFc microdeletion of the Y chromosome.
We report here on the meiotic study of three infertile patients showing classic, complete AZFc microdeletion and severe spermatogenic failure. We combined histological evaluation with immunofluorescence of the first meiotic prophase using anti-SCP3, anti-BRCA1 and anti-H2AX antibodies in the three patients. Meiotic study showed an homogeneous phenotype for the three patients, with a prevalence of synaptonemal complex (SC) fragmentation, dotted SCs and short asynapsis. Mechanisms of spermatogenic failure in AZFc microdeletion are discussed.
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Materials and Methods |
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Patients and controls
This study was performed on three patients (TeH 194, TeH 206 and TeH 220) carrying AZFc microdeletion of the Y chromosome. Patients TeH 194 and TeH 206 consulted in the reproduction center of Marseille, patient TeH 220 consulted in the St Etienne hospital. They were 25, 28 and 41 years of age when they consulted. Patients TeH 194 and TeH 206 presented no personal and familial history of infertility or significant illness. Patient TeH 220 had a right testicular torsion at 16 years of age, and had taken testosterone during several months (bodybuilding). All patients presented normal phenotype, and normal testicular volume. Patients TeH 194 and TeH 206 showed severe oligozoospermia (0.07 x 106 sperm/ml and 0.015 x 106 sperm/ml, respectively). Patient TeH 220 was azoospermic. Patient TeH 194 had a normal FSH level (9.4 IU/ml); patients TeH 206 and TeH 220 had high FSH levels (13.65 IU/ml and 20.93 IU/ml, respectively). The three patients had normal karyotype. All patients had a classic AZFc microdeletion. Two of them (TeH 194 and TeH 206) were deleted from markers sY148 to sY254. Patient TeH 220 was deleted from sY1192 to sY157. All patients underwent testicular biopsy for sperm retrieval before ICSI. A fragment was taken for histopathology study. No spermatozoa for ICSI were found in the testicular fragments of patients TeH 206 and TeH 220. A few spermatozoa were retrieved in the testicular fragments of patient TeH 194, all were immotile after freezing–thawing and ICSI was not performed.
Controls were three patients who showed obstructive infertility. They were selected on the basis of normal FSH and testicular histology, abundant spermatozoa in the testis, and numerous and good quality meiotic cells.
Informed consent of patients was obtained for meiotic studies, and this protocol was approved by the Institutional Ethics Committee.
Immunocytochemistry
Meiotic cells were spread by cytocentrifugation according to the technique described by Metzler-Guillemain and Guichaoua (2000) to perform immunocytochemical analysis. Dual color immunocytochemistry was done with the following antibodies: rabbit polyclonal anti-SCP3 antibody (kind gift of Christa Heyting), a mouse monoclonal anti-H2AX antibody (Euromedex) and a mouse monoclonal (MS110) anti-BRCA1 antibody (ABCAM, Cambridge) to reveal the non-paired XY chromosomes at pachytene stage.
Dual color immunocytochemistry SCP3/H2AX
Cell suspension was fixed before being spread in 0.1% paraformaldehyde. Slides were incubated with anti-H2AX at a dilution of 1/200 for 4 h at 37°C, anti-SCP3 was added on the same preparation at a dilution of 1/200 (Lammers et al., 1994), and the two antibodies were placed overnight in a moist chamber at 37°C. After washes (photoflo–phosphate-buffered saline (PBS)–triton), detection was performed with two secondary antibodies (Zymed, San Francisco, CA). The preparations were incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG at a dilution of 1/200; after washes in photoflo–PBS, the tetramethyl rhodamine B isothiocyanate (TRITC)-conjugated rabbit anti-mouse antibody (1/200) was added on the preparation and incubated for 1 h. Slides were rinsed in PBS and mounted in Vectashield (Vector Laboratories).
Dual color immunocytochemistry SCP3/BRCA1
After spreading, slides were incubated with anti-BRCA1 at a dilution of 1/10 overnight at 37°C, anti-SCP3 was added on the same preparation, and the slides were incubated for 2 h at 37°C. Detection was performed with two secondary antibodies (Zymed, San Francisco, CA). The preparations were first incubated for 1 h with FITC-conjugated goat anti-mouse IgG at a dilution of 1/30, after a wash in photoflo–PBS, the TRITC-conjugated goat anti-rabbit antibody (1/200) was added to the preparation and incubated for 1 h. After washes, the slides were left to dry and were mounted in Vectashield (Vector Laboratories).
Microscopic analysis
Spermatocyte nuclei were observed using a Zeiss Axioplan 2 fluorescent photomicroscope (Zeiss, Germany).
Dual color immunocytochemistry SCP3/H2AX
Two hundred nuclei at leptotene (Fig. 1A), zygotene and pachytene stages were analysed in all patients and controls. For each patient, we evaluated the percentages of the three stages based on the staining of the axial and lateral elements of the SC with SCP3. Nuclei were identified as zygotene on the basis of either discontinuous axial elements and short SC or the ‘bouquet’ configuration (Fig. 1B). For the pachytene nuclei, we evaluated the percentages of the three types of meiotic abnormalities: fragmented SC (Fig. 1C), asynapsed SC (Fig. 1D) and dotted SC, defined as regular discontinuities of SC (Fig. 1E). According to these abnormalities, the nuclei were termed asynapsed nuclei, fragmented nuclei, or dotted nuclei. We defined the four types of H2AX pattern as previously described: absence of staining, diffuse staining, patchy staining and XY-body-reduced staining (Roig et al., 2004).
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Dual color immunocytochemistry SCP3/BRCA1
Hundred pachytene nuclei for each patient and control were classified into four substages (I, II, III and IV) according to the configuration of the XY body revealed with BRCA1 (Guichaoua et al., 2005
). Substages I + II corresponded to early pachytene nuclei, substages III + IV corresponded to late pachytene nuclei.
Statistical analysis
Statistical analysis was performed using the Chi-square test.
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Results |
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Testicular histopathology
In all patients, testicular histopathology showed severe impairment of spermatogenesis. All patients showed diffuse tubular hyalinization, which was very pronounced in patients TeH 194 and TeH 206, with disappearance of tubular lumen, Sertoli and germ cells in some tubules. Sertoli-cell-only tubules were present in patient TeH 206. Severe hypospermatogenesis was present in all other tubules of the three patients. Quantitative evaluation of the seminiferous epithelium, performed on 20 cross-sectioned tubules (Table 1), showed that the mean number of Sertoli cells was normal in the three patients whereas the mean number of germ cells was greatly diminished at all stages, but particularly at spermatid stage. Indeed, the seminiferous tubules contained few or no spermatids, a constant meiotic arrest in pachytene. Some tubules showed sloughing of primary spermatocytes and spermatids.
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Immunocytochemistry
Dual color immunocytochemistry SCP3/
H2AXThe mean percentages of leptotene, zygotene and pachytene nuclei identified with SCP3 were respectively 9, 10.8 and 80.2%, in the three patients, and 5.3, 0.2 and 94.5% in the three controls (Table 2). Patients had significantly more leptotene stages (P < 0.05) and zygotene stages (P < 0.001) than controls, but fewer pachytene stages (P < 0.001). Most zygotene nuclei showed a ‘bouquet’ configuration.
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The mean percentage of abnormal pachytene nuclei was significantly higher in patients (77.3%) than in controls (30.8%) (P < 0.001). The same meiotic abnormalities (asynapsis, fragmented SC and dotted SC) were found in both patients and controls but with various percentages (Table 3). Two or three abnormalities could be associated in the same nuclei. The most frequent abnormality in patients and controls was fragmented SC, isolated or associated with asynapsis and/or dotted bivalents. SC fragmentation varied from a few bivalents per nucleus to all bivalents. The mean percentage of pachytene nuclei with fragmented SC was 46.8% in patients (43.9, 68 and 31.2% in patients TeH 194, TeH 220 and TeH 206, respectively), and 20.8% in controls. The difference between the two groups was significant (P < 0.001). The mean percentage of pachytene nuclei with dotted SC was 31.6% in patients (32.5, 41.2 and 22.7% in patients TeH 194, TeH 220 and TeH 206, respectively) and 4.9% in controls; the difference between the two groups was significant (P < 0.001). As for SC fragmentation, this abnormality varied from a few bivalents per nucleus to all bivalents. The mean percentage of pachytene nuclei with asynapsed SC was 27.6% in patients (30.5, 25.6, and 26.7% in patients TeH 194, TeH 220 and TeH 206, respectively), and 9.5% in controls; the difference between the two groups was significant (P < 0.001). Asynapsis was the least frequent abnormality in patients TeH194 and TeH220 and in pooled data, but it was more frequent than dotted SC in patient TeH206. In the three patients, the asynapsis were short and limited to a few bivalents in most nuclei. Few nuclei showed extended or total asynapsis [5 (3.18%), 0 and 3 (2.02%) pachytene nuclei in patients TeH 194, TeH 206 and TeH 220, respectively].
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The
H2AX pattern for patients and controls showed that H2AX was normally phosphorylated in the two groups. In leptotene stage, nuclei showed strong overall staining; few leptotene cells were not stained (<15%) in the two groups. At zygotene stage, staining was limited to some ‘patches’. In morphologically normal pachytene at substages I, II and III, strong staining was restricted to the XY area; no staining was seen on the substage IV pachytene nuclei in the patients and controls.
Dual color immunocytochemistry SCP3/BRCA1
After BRCA1 staining, XY configuration during pachytene stage was easy to analyse. For patients and controls, we evaluated the percentage of the four pachytene substages (I, II, III and IV) (Fig. 2A–D), and the percentage of early pachytene substages (I + II) and late pachytene substages (III + IV) (Table 4). In patients, the mean percentage of early pachytene nuclei (70.7%) was significantly higher than that of late pachytene nuclei (substages III + IV) (29.3%) (P < 0.001). In the three controls, the number of early pachytene nuclei (32.6%) was significantly lower than that of late pachytene nuclei (67.3%) (P < 0.001).
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Discussion |
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The severity of spermatogenic failure in AZFc-microdeleted patients varies from one patient to another, and in rare cases, men with AZFc microdeletions can father a child. In this study, the three patients bearing complete AZFc microdeletion consulted for primary infertility and had nearly the same testicular histopathological phenotype (Table 1). The meiotic preparations showed few germ cells and the meiotic profile was the same for the three patients. The meiotic picture in these AZFc-deleted patients is fundamentally different from that observed in an AZFb-deleted patient. One difference between meiosis of AZFb- and AZFc- deleted patients is the density profile of meiotic cells. In an AZFb-deleted patient, we previously found a high density of meiotic cells up to substage II pachytene spermatocytes, with, in particular, numerous leptotene nuclei; only two pachytene cells passed the pachytene checkpoint and showed a substage III XY bivalent (Perrin et al., 2006
). Here, AZFc-deleted patients showed few meiotic cells and although the percentage of leptotene spermatocytes was significantly higher than that of controls (Table 2), it was lower than in AZFb deletion (9% versus 21.6%). A particularity of the three AZFc-deleted patients is the higher number of zygotene stages (m = 10.8%), most of them having a ‘bouquet’ configuration, which is a very transitory stage in normal human meiosis (Zickler and Kleckner, 1998). Another difference between the meiosis of the two types of microdeletions was the profile of meiotic abnormalities. In AZFb, the most striking abnormality was an exceptionally high level of extended or total asynapsis, whereas, for the three AZFc-deleted patients, bivalent fragmentation prevailed on asynapsis (Table 3). Moreover, in the three AZFc-deleted patients, asynapsis were limited to short regions and to a few bivalents in the majority of pachytene nuclei analyzed; totally asynapsed nuclei were rarely found.
Although all Y genes are known in the AZFc sequence (Skazletsky et al., 2003; Vogt, 2005), little is known about the spermatogenic functions of these genes. Various approaches have been used to investigate the spermatogenic function of these genes in mice and humans, showing that gene products of the AZFc region have different cellular localizations in testes (Haberman et al., 1998; Reijo et al., 2000; Wong et al., 2004). The present meiotic study brings new information on the chromosomal abnormalities during the first steps of meiotic prophase I, resulting from the absence of AZFc region. In the AZFc region, the gene apparently expressed first is the DAZ gene. Human DAZ protein is present both in fetal gonocytes and in spermatogonia, and to a lesser extent in spermatocytes (Reijo et al., 2000). For CDY2, it has been suggested that this gene is required in the early stages of spermatogenesis (Kleiman et al., 2001). The reduced number of meiotic cells for our patients argues for a weak mitotic activity, which could result from a primary effect of the AZFc microdeletion before meiosis, through the absence of these genes. The AZFc region, could also be critical for the release of the zygotene ‘bouquet’ configuration in pachytene stage. Indeed, in the three patients, the percentage of zygotene stages was significantly higher than that of controls (m = 10.8% versus 0.2%; P < 0.001) (Table 2). We had never observed such a high amount of zygotene stage with bouquet configuration, either in non-obstructive infertility (Guichaoua et al., 2005) or in the patient with AZFb microdeletion (Perrin et al., 2006). Two genes, expressed in spermatocytes, DAZ and VCY2, could be involved in this process. Though the function of VCY2 is still not known, it was suggested it plays a role in the cytoskeletal network via interaction with VCY2IP-1, which could bind to microtubules (Wong et al., 2004). In a detailed analysis of the bouquet stage, Zickler and Kleckner (1998) suggested that chromosome movement at zygotene could be promoted directly via microtubule-mediated ‘yanking’ of chromosome ends. In the present case, the polarization of telomeres occurs normally, but the progressive pachytene dispersal of telomeres around the inner periphery of the nuclear envelope fails, which explains the accumulation of ‘bouquet’ stages in these three patients. The inmost mechanisms of telomere polarization and dispersal during zygotene are different, and it seems that only the second is under the control of the AZFc region. Moreover, Liebe et al., (2003) suggested that a reduction of bivalent condensation would also be consistent with an extended bouquet stage. Thus, the high percentage of dotted SC in the three patients, which we interpret as defective condensation of bivalents, could be related with late zygotene accumulation and could result from the direct effect of AZFc on early prophase. Nevertheless, an extended zygotene stage, also described in mouse Sycp3–/– (Liebe et al., 2003) and in Atm–/– spermatocytes (Scherthan et al., 1996), could be due to apoptosis of spermatocytes, as suggested in these two studies. Although the knockout mouse for H2AX displays a significantly extended bouquet stage (Fernandez-Capetillo et al., 2003), the H2AX labeling at leptotene and pachytene nuclei was normal in our three patients. This observation indicates that the initiation of genetic recombination is normal and is not impaired by AZFc microdeletion. Genes of the AZFc region, such as DAZ (Haberman et al., 1998), CDY1 (Lahn et al., 2002) and VCY2 (Wong et al., 2004) are also expressed out of meiosis, during spermiogenesis, but this stage of spermatogenesis is out of the scope of this study.
Although SC were formed in the three patients, abnormalities other than dotted SC were significantly higher than in controls. Among these SC abnormalities, fragmented SC could result from a secondary effect. Indeed, most spermatocytes that reach the pachytene stage have suffered from the direct effect of the Y microdeletion in the previous stages; chromatin and SC are progressively fragmenting and the cells are degenerating. In the same way, limited asynapsis likely results from a secondary effect: the deleterious effect on germ cells could be secondary to the impaired testicular somatic environment due to the AZFc microdeletion (Guichaoua et al., 2005).
As in two previous studies (Guichaoua et al., 2005; Perrin et al., 2006), staging of the pachytene cells, according to XY chromosome morphology (Speed et al., 1993), revealed a prevalence of early pachytene substages (PI + PII = 70.7%; PIII + PIV = 29.3%) (Table 4). We suggested that the pachytene checkpoint is localized at the mid-pachytene stage in humans, based on the findings that various causes of male infertility result in prevalence of early pachytene substages. The present study shows that most germ cells with AZFc microdeletions also are arrested by the pachytene checkpoint. The severity of the arrest was much greater in AZFb microdeletions than in AZFc. Because the normally synapsed pachytene failed to pass the pachytene checkpoint, also referred to as ‘meiotic recombination checkpoint’, we postulated that a failure of meiotic recombination would be responsible for the spermatogenic failure in patients with AZFb microdeletions (Perrin et al., 2006). The meiotic pictures obtained in the present study, normal phsophorylation of H2AX and short asynapsis, suggest that the mechanisms of spermatogenic failure resulting from AZFc microdeletion are highly different from those of AZFb microdeletion, and do not seem to involve specially the meiotic recombination.
In conclusion, the present study, together with the previous meiotic investigation of an AZFb deletion (Perrin et al., 2006) point out the fundamentally different behavior of chromosomes during the first stages of meiotic prophase for AZFb and AZFc microdeletions. Meiotic studies thus bring interesting information on the mechanisms by which microdeletions impair spermatogenesis and, thereby serve to define the roles of the genes of the AZFb and AZFc regions of the Y chromosome. Although all differences between patients and controls were statistically significant, it should be noted that only three patients and three controls were analysed. Other meiotic investigations, with new meiosis-specific antibodies, and more patients with different breakpoints in AZFb and AZFc regions, are needed to determine the respective roles of these two regions and of their genes in spermatogenesis.
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Acknowledgments |
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The authors thank Prof. C Heyting for her generous gift of SCP3 antibody; Prof. Gamerre, Dr A. Noizet, Dr G. Porcu, Dr F. Carles and Prof. Grillo for their clinical and biological contribution; C. Metton for technical assistance; N Gros and A Clemenson for histopathological analysis. The authors thank Gary Burkhart for correction of the English and the two anonymous reviewers for their constructive comments. This work was supported by grants from Assistance Public of Marseille.
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Submitted on July 3, 2006; resubmitted on January 10, 2007; resubmitted on January 31, 2007; accepted on February 6, 2007.
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