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Sex Chromosome Alignment at Meiosis of Azoospermic Men With Azoospermia Factor Microdeletion

SHMUEL SEGAL

RONNI GAMZU

BATIA B. MAYMON

From ^* The Institute for the Study of Fertility and Obstetrics and Gynecology, Lis Maternity Hospital, and Institute of Pathology, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Israel, and Obstetrics & Gynecology Department, Barzilai Medical Centre, Ashkelon, Israel.

Correspondence to: Leah Yogev, PhD, The Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel (e mail: layogev{at}zahav.net.il).

Received for publication May 20, 2003; accepted for publication July 28, 2003.

	Abstract

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Deletions in the q arm of the Y chromosome result in spermatogenesis impairment. The aim of the present study was to observe the X and Y chromosome alignment in the spermatocytes of men with Y chromosome microdeletion of the azoospermia factor (AZF) region. This was performed by multicolor fluorescence in situ hybridization probes for the centromere and telomere regions. Testicular biopsies were performed in a testicular sperm extraction-intracytoplasmic sperm injection set-up in 11 azoospermic men: 8 (nonobstructive) with AZF deletions and 3 (obstructive) controls. Histological sections, cytology preparations of the testicular biopsies, and evaluation of the meiosis according to the percentage of XY and 18 bivalents formation were assessed. Spermatozoa were identified in at least one location in controls and specimens with AZFc-deleted Y chromosomes. Complete spermatocyte arrest was found in those with a deletion that included the entire AZFb region. Bivalent formation rate of chromosome 18 was high in all samples (81%-99%). In contrast, the rate of bivalent X-Y as determined by centromeric probes was lower but in the range favorable with spermatozoa findings in controls and patients with the AZFc deletion (56%-90%), but not in those with AZFb-c deletions (28%-29%). A dramatic impairment in the normal alignment of X and Y telomeres in the specimen with AZFb-c deletion was shown (29%), compared to the specimens with AZFc deletion (70%-94%). It is suggested that the absence of sperm cells in specimens with the entire AZFb and with AZFb-c deletions is accompanied by meiosis impairment, perhaps as a result of the extent of the deletion or because of the absence of genes that are involved in the X and Y chromosome alignment.

     Key words: Azoospermia, chromosome pairing, FISH, Y chromosome microdeletion

Deletion of the entire AZFa region (0.8 Mb; Sun et al, 2000) is associated with lack of germ cells (Sertoli-cell-only syndrome), and complete deletion of the AZFb region (6.23 Mb; Repping et al, 2002) is generally associated with arrest of spermatogenesis (Krausz et al, 2000; Foresta et al, 2001; Kleiman et al, 2001; Luetjens et al, 2002). Deletion of AZFc is the most common, and is associated either with severe oligozoospermia, or azoospermia, accompanied by a wide spectrum of histological defects (Reijo et al, 1995, 1996). It is remarkably uniform, spanning a 3.5-Mb segment (Kuroda-Kawaguchi et al, 2001).

Both AZFc and AZFa deletions, as well as most AZFb deletions, appear to ensue from homologous recombination between direct repeats on the Y chromosome (Kamp et al, 2000; Sun et al, 2000; Kuroda-Kawaguchi et al, 2001; Repping et al, 2002). Yq microdeletions may be associated with Y chromosomal instability, leading to the formation of the 45,X0 cell in lymphocytes and sperm cell nullisomic for the Y chromosome (Siffroi et al, 2000). The use of sperm retrieval from testes of infertile men to achieve fertilization through intracytoplasmic sperm injection (ICSI) raises the possibility of transmission of Y-related infertility to the male offspring (Kent-First et al, 1996; Vogt et al, 1996; Kleiman et al, 1999b).

During late zygotene/early pachytene stages of the first meiotic division, X and Y chromosomes form a special structure, known as the sex vesicle (Pathak and Elder, 1980) or the XY body (Solari, 1999), which is characterized by a particular chromatin condensation and transcriptional inactivation. The special chromatin condensation of the XY body during meiotic prophase is thought to serve to prevent accidental recombination events between nonhomologous regions of the X and Y chromosomes (Handel and Hunt, 1992; Mckee and Handel, 1993). In the XY body structure chromosomes X and Y are organized into 2 completely separate chromosomal domains (Metzler-Guillemain et al, 2000). Despite the separation, the ends of the X and Y chromosome domains are joined by their pseudoautosomal regions, as was demonstrated by short synaptonemal complex formation (Pathak and Elder, 1980) and by the clustering of their 4 telomeres (Metzler-Guillemain et al, 2000).

The length of the synaptic region varies from cell to cell (Pathak and Elder, 1980). It is not limited to the pseudoautosomal region, and can include a significant portion of the Y chromosome, leading to apparent nonhomologous pairing (Chandley et al, 1984). However, normal sequence exchange occurs exclusively within two stretches of homology, and is most prevalent within the 2.5 Mb of DNA, adjacent to the short arm telomeres of both the X and Y chromosomes.

Crossing within the pseudoautosomal region is functionally significant in that it generates the chiasma that is required to hold the sex chromosomes together during the first metaphase of meiosis. Frequency of recombination within the long arm pseudoautosomal region (320 kb) is high; however, it is neither necessary nor sufficient for successful meiosis (Freije et al, 1992; Kvaløy et al, 1994). Data are most consistent that X-Y pairing and recombination are necessary for a spermatocyte to successfully complete meiosis (McIlree et al, 1966; Chandley and Edmond, 1971; Gabriel-Robez et al, 1990; Hale, 1994).

The fluorescence in situ hybridization (FISH) technique can be used for bivalent evaluation with a certain advantage over other techniques. This is mainly because there is no need for microspreading as a precondition (Scherthan et al, 1996). Thus, it can facilitate the evaluation of hundreds of meiotic cells from each specimen, even in pathological cases with an extremely low number of spermatocytes, reflecting serious testicular damage. In contrast, an alternative method, such as microspreading followed by synaptonemal complex protein staining, which is the most common technique for the study of meiotic pairing, is less applicable in such extreme cases (Metzler-Guillemain and Guichaoua, 2000).

In a recent study using testicular biopsies of azoospermic men, a significantly higher rate of some homologous chromosome bivalents was found whenever spermatozoa were detected. Bivalent formation of all 4 pairs of chromosomes that were evaluated was highly correlated, but the rate of the bivalent X-Y was found to be the most sensitive predictor for detection of spermatozoa, with a cutoff value of 47% (Yogev et al, 2000; Yogev et al, 2002).

The purpose of the present study was to observe the X and Y alignment in spermatocytes of men carrying Y chromosome microdeletions. This was performed to evaluate whether the AZF-deleted segment may influence the ability of the Y chromosome to pair. Multicolor FISH probes for both centromere and telomere regions were used. Evaluation of XY bivalent in specimens with AZF microdeletion has not been reported previously.

	Materials and Methods

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A total of 11 azoospermic men undergoing testicular sperm extraction (TESE) were enrolled in the study. In all cases, azoospermia was reconfirmed before the TESE procedure. The etiology of four men was AZFc deletion (between direct repeats b2/b4): two had AZFb (between palindromes P5/proximal P1) and two others had both AZFb and AZFc deletions [between palindromes P5/distal P1 and P5/del(qter), Table 1]. Three men with obstructive azoospermia due to congenital absence of vas deferens (carriers of cystic fibrosis mutations) served as a control group. Partial evaluation of three cases (nos. 1, 2, 9) was previously published (Yogev et al, 2002).

Testicular volume was between 8 and 25 mL. Four men (nos. 1, 3, 4, 5) had elevated follicle-stimulating hormone levels (>11 mIU/mL). All patients provided written informed consent to undergo genetic evaluation. The study was approved by the local Institutional Review Board Committee in accordance with the Helsinki Declaration of 1975. The evaluation included karyotyping, Y-chromosome microdeletion test, and the percentage of homologous chromosome pairing in spermatocytes. Chromosome karyotyping was performed on peripheral lymphocytes with G-banding. The Y-chromosome microdeletion test was performed by the multiplex polymerase chain reaction with genomic DNA isolation from peripheral blood samples, as previously described (Kleiman et al, 1999a). Presence of the Yq-tip was verified with marker sY1201 (Kuroda-Kawaguchi et al, 2001). The deletion boundaries were determined using additional markers (sY1207; sY1228; sY118; sY1224; sY1192; sY1291; sY639; sY1257).

Biopsy Evaluation

Details of the TESE procedure have been described previously (Hauser et al, 1998; Yogev et al, 2000). Briefly, one biopsy specimen obtained from each testis was divided into two: one small section (<20mg) was utilized for routine Bouins (Sigma Chemical Company, St. Louis, Mo) fixation and histopathologic assessment, counting of testicular cells, and pairing evaluation by FISH. The other section (approximately 50 mg) was minced for spermatozoa extraction to be used in the ICSI procedure. Two additional biopsy specimens were procured from each testis for spermatozoa extraction, with the exception of 2 clearly defined, obstructive, azoospermic patients (Table 1, nos. 9, 11).

Histological analysis of spermatogenesis was performed on hematoxylin- and eosin-stained sections. At least 20 seminiferous tubules were scored in each testis. The number of Sertoli and germ cells at different stages was counted for each tubule section, and the mean value was calculated. The same technician viewed all the slides.

Specimen Preparation and Detection of Chromosome Pairing

Testicular biopsy specimens were prepared as previously described (Yogev et al, 2000). Briefly, mechanical minced tissue was fixed with cold fresh methanol/acetic acid (3/1) solution, dropped onto 2 slides (Super Frost/plus; Menzel-Glaser, Braun-schweig, Germany), and air dried. All the different testicular nuclei, including spermatozoa, were counted. The percentage of spermatocytes and spermatozoa in the sample was calculated from approximately 1600 testicular nuclei per sample.

For detection of chromosome pairing by FISH, the slides (two for each sample) were treated according to the manufacturer's instructions. Triple-color FISH with CEP centromeric DNA probes (Vysis, Downers Grove, Ill) for chromosomes X (spectrum green), Y (spectrum orange), and 18 (spectrum aqua blue), and double-color FISH with TelVysion telomeric probes Xq/Yq (spectrum orange) and Xp/Yp (spectrum green) were used. Two independent observers viewed each slide with an Olympus AX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a single band for 4',6-diamidino-2-phenylindole (DAPI), triple-band pass filter for DAPI/fluorescein isothiocyanate (FITC)/TRIC, and a single-band pass filter for FITC and aqua blue.

Nuclei of primary spermatocytes were identified by the size of the nucleus in comparison to the other nuclei, and by the typical granular or threadlike chromatin appearance of the DAPI counterstain. Generally, at least 350 spermatocytes were analyzed in each slide. Cells were scored according to the number and proximity of the signals. The scored nuclei were intact and did not overlap other nuclei. For X and Y chromosomes, only centromeric signals with a maximum of two-signal diameters apart were recognized as close bivalents and marked as X-Y. For the autosomal 18 chromosome bivalent, when one signal, or two signals of similar size, were located at a distance of less than one diameter of the signal domain, a paired bivalent was recorded. For the telomere signals, pairing of the short and long arms of X and Y chromosomes was determined by the same criteria as those for the autosomal 18 chromosome bivalent. The percentage of pairing was calculated by dividing the number of nuclei with paired bivalents by the number of spermatocytes that were evaluated in each slide. The reproducibility between the score of the 2 technicians was <5% of variance (r = 0.95 by Spearman test).

Statistical Analysis

Variances between pairing of p and q X and Y chromosomes were analyzed using the two-tailed Wilcoxon signed rank test. Correlation was evaluated using the Spearman correlation test. All statistical analyses were performed using SPSS for Windows 9.0 (SPSS Inc., Chicago, Ill).

	Results

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In all 4 patients with AZFc deletion (Table 1), spermatozoa were identified in at least 1 of the 6 locations that had been evaluated (Figure 1b). In the testes of the 2 patients with AZFb deletion and the 2 with AZFb-c deletion, no spermatozoa could be found (Figure 1c and d). In the histological section, which represents only 1 location in each testis, mature spermatids were found in only 2 patients with AZFc deletion (Table 2, nos. 3 and 4).

View larger version (114K): [in this window] [in a new window]   Figure 1. Testicular histology and chromosome location in the spermatocyte nucleus. (a) Normal spermatogenesis (control, patient no. 10); (b) mixed atrophy (AZFc deletion, patient no. 3); (c) spermatocyte arrest (AZFb deletion, patient no. 5); (d) spermatocyte arrest (AZFb-c deletion, patient no. 7). Scale bar = 50µm. (e-h) Green and orange signals for X and Y centromeres, respectively. Aqua blue color signal for 18 chromosome centromere. Chromosome status as bivalents are presented: X and Y in proximity (e, g), one signal for the two-paired 18 homologous (e, f), and univalency for X, Y (f, h) and the two 18 chromosomes (g, h). (i-l) Green and orange for p- and q-arms' telomeres of both X and Y chromosomes, respectively. One signal for paired X and Y p-arms (i-k), one signal (i) or two closed signals (j) for both X and Y q-arm telomeres, and univalency for p-arms (l) and q-arms (k, l) of X and Y chromosomes. Scale bar = 10µm.

In the suspension of minced testicular tissue stained by the FISH technique, no spermatozoa were found in any of the samples of AZF-deleted Y chromosomes (Table 2, nos. 1 through 4, and 7 and 8). The percentage of spermatocytes (calculated from the testicular nuclei) varied between 0% and 3% and 0.1% and 9% in men with AZFc and AZFb-c deletions, respectively. The absence of spermatocytes in specimen no. 4 and specimens for FISH in patients 5 and 6 prevented their meiosis evaluation.

All patients had normal karyotype, except for patient no. 8 who was 46,XYqh-/45,X (82%/18%). In this specimen, telomere pairing was not shown because of being not informative.

A total of 7623 spermatocytes were evaluated. Pairing rate of chromosome 18 (Figure 1e and f) was high in all samples (Table 3). Despite this high rate of pairing, previously found to correlate with the presence of spermatozoa (Yogev et al, 2000; Yogev et al, 2002), only spermatocytes were found as the most advanced stage in the biopsies of patients with AZFb-c deletion. Nevertheless, using the X-Y centromeric probes, the rate of bivalent X-Y (X and Y in proximity, Figure 1e and g) was under the cutoff value of 47%, indicating the lack of spermatozoa in patients with the AZFb-c deletions (nos. 7 and 8). The rate of bivalent X-Y was above the cutoff value in biopsies of patients with AZFc deletion (Table 3).

Interestingly, the rate of pairing of chromosomes X and Y in the p arms (Figure 1i through k) was higher than the percentage of spermatocytes with adjacent centromeres for all specimens (P = 0.0225). However, when a high percentage of spermatocytes with the X and Y q-arms far apart was recognized (as was in no. 7, Figure 1k and l), a distance was also found between the centromeric signals. No significant correlation was found between pand q-arms rate of pairing, whereas the highest value of correlation was found between the centromeres of X-Y in proximity and paired q-arms (Table 4).

	Discussion

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It is well accepted that the region and extent of the Y chromosome microdeletion play a major role in the magnitude of spermatogenesis impairment (Kleiman et al, 2001; Luetjens et al, 2002). It is also known that failure of adjacency between the X and Y chromosomes generates complete breakdown of meiosis (Hale, 1994). In the present study, as was shown also by Krausz et al, no mature sperm cells were detected when the deletions included the entire AZFb or AZFb-c regions (Krausz et al, 2000).

Pairing impairment was found despite the fact that the deletion extended away from the pseudoautosomal region of the q-arm, to the AZFb domain. This finding hints at 2 probable theories: The first is that the extent of the deletion is responsible for the pairing impairment, by generating a spatial disturbance. The second possibility is that the gene/s in AZFb region are accountable for the process of X-Y bivalent formation during meiosis (18-18 pairing was close to normal).

AZFb deletion, encompassing up to 6.2 Mb, eliminates 32 genes and transcripts and AZFb-c deletion comprehends 7-7.7 Mb. This is in contrast to the 3.5 Mb of the AZFc region. The deletions exclude members of testis-specific families of genes that were proposed to be involved in the spermatogenic process (Repping et al, 2002). Only the heterochromatic region, which is variable in its size (about 40 Mb in a study of one man) is in between the AZFc and the pseudoautosomal region (Skaletsky et al, 2003). Obviously, further studies of additional patients with isolated AZFb deletion may verify the correct hypothesis for the spermatogenesis impairment. Since only one autosomal bivalent formation (chromosome 18) was explored, pairing status of some other autosomes may help to confirm the difference between the X-Y bivalent in contrast with the autosome rate of pairing.

It is commonly acclaimed that large microdeletions that include the heterochromatic Yq tip ('terminal' deletion) may cause chromosomal instability (Siffroi et al, 2000). This phenomenon can explain the karyotype of patient no. 8, who was found to be mosaic 46,XYqh-/45,X (82%/18%). Surprisingly, the rate of X-Y bivalents was similar for the 2 men with AZFb-c deletion, although specimen no. 8 had a larger deletion that included the Yq tip. Nevertheless, the number of germ cells in the testicular biopsy of patient no. 8 (Table 2) was lower compared with those of patient no 7.

In normal pairing of X and Y chromosomes, there was clustering of their 4 telomeres in such a way that in 90% of the spermatocytes, only 2 juxtaposed signals were detected (Metzler-Guillemain et al, 2000). In our study, 95%-96% of the spermatocytes in the control specimens were found with all signals close to each other. Whereas AZFc deletion was found to be associated with a moderate decrease in the frequency of this phenomenon (70%-94%), the specimen with AZFb-c deletion showed a dramatic impairment (29%). Therefore, the low rate of clustering of short and long arms of both X and Y telomeres may indicate for the dramatic impairment of the meiosis.

As shown previously, when no spermatozoa or mature spermatids were found in some biopsies, the relatively high rate of the bivalents indicated the possibility of finding some foci of spermatogenesis in other biopsies. Failure in X-Y bivalent formation appears to lead to breakdown of spermatogenesis at the time of the meiotic division (Yogev et al, 2002). Interestingly, in the patient with AZFb-c deletion (no. 7), the short arms paired in 51% of the spermatocytes. However, despite quite a high level of X-Y short-arm pairing, the typical XY body chromatin probably was not formed and consequently, the rate of the detected X-Y bivalent that was recorded by centromeres in proximity was 29% only. A different finding was reported for the short arm of the X chromosome in 2 men with a karyotype of 46,Y,der(x),t(X; Y)(p22.3:q11). Analysis of synaptonemal complexes at pachytene showed that deletion of the X short-arm pseudoautosomal region in these men impaired sex-chromosome pairing even though a "sex vesicle" was formed (Gabriel-Robez et al, 1990).

It can therefore be suggested that, in contrast to the AZFc deletion, the AZFb- and AZFb-c-deleted regions may cause a meiotic impairment by disturbing the X and Y chromosome alignment. Obviously this finding has to be confirmed by increasing the number of patients. The evaluation of the XY body, using monoclonal antibodies against its specific proteins, may assist in clarifying the nature of the meiosis impairment. Our results support the concept that the analysis of Y chromosome microdeletion is of clinical importance not only in terms of defining the etiology of the spermatogenesis impairment, but also because of its clinical prognostic value.

	Acknowledgments

 
We thank Mrs Bella Gore, Marina Ilatov, and Jenny Yerushalmi for their excellent technical assistance. The support of the entire staff of the Institute for the Study of Fertility is much appreciated.

	Footnotes

 
Supported by grants from the Chief Scientific Office, Ministry of Health, and by the Hirsch and Genia Wassermann Memorial Fund for Medical Research, Tel Aviv University.

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