Unique t(Y;1)(q12;q12) reciprocal translocation with loss of the heterochromatic region of chromosome 1 in a male with azoospermia due to meiotic arrest: a case report

Maria João Pinho1, Rui Neves1, Paula Costa1, Cristina Ferrás1, Mário Sousa1,2,3,4, Cláudia Alves1, Carolina Almeida1, Susana Fernandes1, Joaquina Silva2, Luís Ferrás2 and Alberto Barros1,2

1 Department of Genetics, Faculty of Medicine, 2 Centre for Reproductive Genetics A.Barros and 3 Laboratory of Cell Biology, ICBAS, University of Porto, Portugal

4 To whom correspondence should be addressed at: Laboratory of Cell Biology, ICBAS, University of Porto, Lg Prof Abel Salazar 2, 4099-003 Porto, Portugal. Email: msousa{at}icbas.up.pt


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A de novo reciprocal translocation 46,X,t(Y;1)(q12;q12) was found in an azoospermic male with meiotic arrest. Cytogenetics and fluorescent in situ hybridization (FISH) were used to define the karyotype, translocation breakpoints and homologue pairing. SRY (Yp), Yq11.2-AZF regions, DAZ gene copies and the distal Yq12 heterochromatin were studied by PCR and restriction analysis using sequence-tagged sites and single nucleotide variants. High resolution GTL, CBL and DA-DAPI staining revealed a (Y;1) translocation in all metaphases and a normal karyotype in the patient's father. FISH showed the presence of the distal Yq12 heterochromatic region in der(1) and loss of the heterochromatic region of chromosome 1. PCR demonstrated the intactness of the Y chromosome, including the SRY locus, AZF regions, DAZ genes and distal heterochromatin. A significant decrease (P=0.005) of Xp/Yp pairing (18.6%), as compared with controls (65.7%), was found in arrested primary spermatocytes, and cell culture and mRNA expression studies confirmed an irreversible arrest at meiosis I, with induction of apoptosis and removal of germ cells by Sertoli cells. We characterized a de novo t(Y;1)(q12;q12) balanced reciprocal translocation with loss of the heterochromatic region of chromosome 1, that caused unpairing of sex chromosomes followed by meiosis I arrest, apoptotic degeneration of germ cells and azoospermia.

Key words: apoptosis/AZF/DAZ/meiosis I arrest/t(Y;1)(q12;q12) de novo balanced reciprocal translocation/Y chromosome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In mammals, the Y chromosome is essential for sex determination, early sexual differentiation and control of spermatogenesis (Disteche et al., 1986Go; Arnemann et al., 1991Go; Gardner and Sutherland, 1996Go). In the general population, the incidence of Y–autosome translocations is ~1:2000 (Nielsen and Rasmussen, 1976Go; Gardner and Sutherland, 1996Go; Powell, 1999Go). In particular, translocations involving the Y and the non-acrocentric chromosomes are even more rare and may involve any segment of the Y chromosome (Pajares et al., 1979Go; Smith et al., 1979Go). It has been assumed that males are fertile when the breakpoint locates in the heterochromatic region of Yq12, whereas male infertility occurs when the breakpoint lies in the region of the azoospermia factor (AZF) locus at Yq11 (Vogt and Fernandes, 2003Go). However, some reported cases showed that breakpoints in the Yq12 heterochromatic region may be associated with male infertility (Hsu, 1994Go; Delobel et al., 1998Go) and that breakpoints in the Yq11 euchromatic region may also occur in fertile males (Teyssier et al., 1993Go). Apart from some exceptions associated with fertile or subfertile phenotypes (Hsu, 1994Go; Giltay et al., 1999Go), Y–autosome translocations usually lead to male infertility. Although abnormal phenotypes other than infertility have also been ascribed to Y–autosome translocations (Mattei et al., 1978Go; Moreau et al., 1987Go; Hsu, 1994Go), these are considered to represent casual but not causative events (Nielsen and Rasmussen, 1976Go; Moreau et al., 1987Go; Morel et al., 2002Go).

To the best of our knowledge, five cases of balanced reciprocal (Y;1) translocations in adult males have been published previously. In all these cases, patients presented with infertility but the breakpoints were not at q12 in both chromosomes (Hsu, 1994Go; Maraschio et al., 1994Go; Pabst et al., 2002Go). We report here the results of cytogenetic and molecular studies carried out in an azoospermic male showing a de novo balanced reciprocal 46,X,t(Y;1)(q12;q12) translocation with loss of the heterochromatic region of the translocated 1q12 region, which caused spermatogenic arrest at meiosis I. Similar breakpoints but without loss of the heterochromatic region of chromosome 1 have been reported previously in patients with malignant haematological disorders (Michaux et al., 1996Go). Thus, the present case corresponds to a new subtype of t(Y;1) translocation and the first described in a patient with infertility as the only phenotypic abnormality.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Informed consent was obtained from the patient and his relatives before inclusion in the study.

Karyotyping and fluorescence in situ hybridization
High-resolution chromosomal GTL, CBL and DA-DAPI bandings were performed on cultured, phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes, according to conventional cytogenetic procedures (Verma and Babu, 1995Go). Fluorescence in situ hybridization (FISH) was performed as previously described (Alves et al., 2002Go), using {alpha}-satellite probes for the centromeric regions of chromosomes X (DXZ1, Xp11.1–q11.1; Vysis Inc., Downers Grove, IL), Y (DYZ3, Yp11.1-q11.1; Vysis) and 1 (D1Z5, 1p11.1–q11.1; this probe also hybridizes with chromosomes 5 and 19; Cytocell, Oxfordshire, UK), satellite III probes for the heterochromatic regions of chromosomes Y (DYZ1, Yq12; Vysis) and 1 (D1Z1, 1q12; Cytocell), an LSI probe for the Y chromosome SRY locus (SRY, Yp11.3; Vysis) and subtelomeric probes for the X and Y short arms (TelVysion Xp/Yp, DXYS129; Vysis), and for the long arms of chromosomes 1 (tel 1q, D1S3739; Cytocell), X and Y (TelVysion Xq/Yq, Z43206; Vysis). Normal male lymphocytes were used as controls. After rinsing, slides were dehydrated, air dried and mounted in 10 µl of Vectashield antifade medium containing 1.5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei (Vector Laboratories, Burlingame, CA). Images were recorded in a Nikon (Eclipse, E-400; Tokyo, Japan) epifluorescence microscope fitted with a CCD camera (Sony, Tokyo, Japan) and appropriate software (Cytovision Ultra, Applied Imaging International, Sunderland, UK).

PCR amplification for SRY and AZF regions
Peripheral blood (8–10 ml) was pelleted and stored at –20°C until DNA extraction. Genomic, high molecular weight DNA was isolated using a salting-out method. Yq11.2-AZF microdeletions were screened by multiplex PCR using 15 sequence-tagged sites (STSs) (Laboratorial licence from EQAS Y Chromosome, 2002): AZFa: sY84, USP9Y, GY6 (DBY); AZFb: sY691 (EIF1AY), sY134, sY135, sY142; and AZFc: BPY2 (BPY2), sY152 (DAZ), sY254 (DAZ), DAZ1 (DAZ), sY157, CDY1 (CDY). sY14 (SRY) and TSPY (TSPY) were used as positive internal controls on the multiplex reactions. Genomic DNA samples of fertile men and normal females were used, respectively, as positive and negative controls in each PCR experiment (Fernandes et al., 2002Go).

DAZ gene copy-specific deletion analysis
As no microdeletions were found in the Yq11.2-AZF regions, a DAZ gene copy deletion analysis was performed using six DAZ single nucleotide variants (SNV I–VI) and two STSs (DAZ-RRM3, Y-DAZ3) as previously described (Fernandes et al., 2002Go; Ferrás et al., 2004Go). Briefly, alleles A and B in SNV I code for the integrity of proximal DAZ4, allele A in SNV II for DAZ1, allele A in SNV III for proximal DAZ2 and in SNV IV for distal DAZ2, allele A in SNV V for DAZ3 and DAZ4, allele B in SNV V for DAZ1 and DAZ2, allele B in SNV VI for distal DAZ4, RRM3 for DAZ1 and DAZ4, DAZ3 for DAZ3, and sY152 for DAZ1 and DAZ4. Controls were as above.

PCR amplification of distal Yq12 heterochromatin
Three STSs (sY160, sY1124 and sY1245; GenBank accession numbers: G38343, G66138 and G75491) were used to study the distal Y chromosome heterochromatic region. PCRs were performed in 25 µl reaction volumes with 2.5 µl of 10x buffer (100 mmol/l Tris–HCl, pH 8.3, 500 mmol/l KCl; MBI Fermentas, St Leon-Rot, Germany), 0.75 µl of MgCl2 (25 mmol/l; MBI Fermentas), 1 µl of dNTP mix (12.5 pmol/µl each dNTP; Invitrogen, Barcelone, Spain), 0.5 µl of each primer pair (12.5 pmol/µl) and 0.2 µl of Taq recombinant DNA polymerase (5 U/µl; MBI Fermentas). Specific PCR conditions were: a pre-soak of 5 min at 95°C and 35 cycles with denaturation for 30 s at 95°C, annealing for 30 s (63°C for sY160, 66°C for sY1124 and sY1245), polymerization for 1 min at 72°C, and a final extension for 5 min at 72°C. PCR products (10 µl aliquots) were analysed on 2.5% agarose gels stained with ethidium bromide. Controls were as above.

Male germ cell isolation and culture
After bilateral testicular biopsy, the seminiferous tubules were digested enzymatically (Sousa et al., 2002bGo). For FISH analysis, germ cells were isolated by micromanipulation in an inverted Nikon microscope, equipped with Hoffman optics and a heated stage (32°C), using Narishige micromanipulators (Nikon, Tokyo, Japan) and micropipettes of 15–20 µm in diameter (SweMed, Frolunda, Sweden). For testing germ cell in vitro differentiation, cell suspensions were cultured in tubes for 2 weeks in Vero cell (Vircell SL, Santa Fe, Granada, Spain) conditioned medium supplemented with 25 IU/l recombinant FSH (Serono, Geneve, Switzerland; Organon, Oss, The Netherlands) and 1 µmol/l water-soluble testosterone (Sigma, Barcelone, Spain) at 32°C, 5% CO2 in humidified air (Cremades et al., 1999Go, 2001Go; Sousa et al., 2002aGo).

Meiotic studies
Sequential FISH was performed (Sousa et al., 2002aGo) using {alpha}-satellite probes (Vysis) for the centromeric regions of chromosomes X (DXZ1, Xp11.1–q11.1), Y (DYZ3, Yp11.1–q11.1), 7 (D7Z1, p11.1–q11.1) and 18 (D18Z1, p11.1–q11.1), a satellite III probe for the heterochromatic region of chromosome Y (DYZ1, Yq12), and a telomeric probe for the X and Y short arms (TelVysion Xp/Yp). As controls for normal homologue meiotic pairing, we used primary spermatocytes isolated from a treatment testicular biopsy of a patient with secondary obstructive azoospermia, normal karyotype and conserved spermatogenesis. Positive signals were obtained in 108 out of 125 of the cells (86.4%) for sex chromosomes and in 115 out of 125 of the cells (92%) for autosomes.

mRNA expression analysis
RNA extraction was performed from testicular cell suspensions with an RNeasy Mini Kit (Qiagen, Hilden, Germany) and converted to cDNA by the SuperScript First-Strand Synthesis System (Invitrogen) using oligo(dT) primers. PCRs were performed for caspases 8, 9 and 3 (Fernandes-Alnemri et al., 1994Go; Teitz et al., 2000Go, 2002Go), and for Fas receptor, Bcl2 and Bax (Sigma). Samples consisted of whole testicular tissue, either fresh or after long-term culture. Peripheral blood lymphocytes from a leukaemia patient at remission were used as a positive control.

Statistics
Proportions were compared using the difference between two proportions test (Statistica, version 5.1), with the significance of the P-value being set at 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A Caucasian 35-year-old man with normal body size, 170 cm in height, ARh+, from the southern region of Portugal was referred to our IVF centre due to infertility of 10 years duration. There were no relevant findings regarding his past history and habits. Both physical examination and ultrasound analysis of the genital–urinary system were normal, with normal testicular volume and normal appearance of the epididymis and vas deferens. The spermiogram, after centrifugation, showed azoospermia, with normal semen volume, pH, fructose and citric acid levels. The haematological, biochemical and serological assays were normal. Hormone serum levels were 6.2 mU/ml FSH (normal = 1.1–13.5), 5.9 mU/ml LH (0.4–5.7), 6.5 ng/ml testosterone (2.7–10.7), 15 pg/ml estradiol (10–44) and 17.6 ng/ml prolactin (3.1–16.5). The bilateral diagnostic testicle biopsy revealed slight thickening of the basal lamina, peritubular fibrosis and Leydig cell hyperplasia, with the seminiferous tubules being filled with Sertoli cells and only a few germ cells at the spermatogonia and primary spermatocyte stage.

In all 30 observed metaphases, GTL banding showed a translocation involving chromosomes Y and 1 (Figure 1A and B). CBL (Figure 1C and D) and DA-DAPI (Figure 1E and F) staining revealed that the derivative chromosome 1, der(1), was composed of the short arm and centromere of chromosome 1, whereas the derivative chromosome Y, der(Y), consisted of the short arm, centromere and heterochromatic region of the Y chromosome, followed by the long arm of chromosome 1. The patient's father had a normal karyotype (46,XY), demonstrating that this was a de novo t(Y;1)(q;q) balanced reciprocal translocation.



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Figure 1. GTL, CBL and DA-DAPI studies. (A and B) GTL, (C and D) CBL and (E and F) DA-DAPI paterns.

 
The combined use of different probes in metaphase FISH experiments confirmed these observations, including the intactness of telomeres, short arms and centromeres, and showed loss of the 1q heterochromatic region and translocation to der(1) of the distal part of the Yq heterochromatic region (Figure 2). This indicates that the breakpoint was in the distal heterochromatic region of the long arm of the Y chromosome (Yq12) and in the proximal heterochromatic region of the long arm of chromosome 1 (1q12).



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Figure 2. Metaphase FISH studies. (A) Y satellite (blue), Y centromeric (green) and Y SRY (red) probes (3 h hybridization). (B) Y satellite (blue) and Y centromeric (red), X centromeric (yellow) and Xp/Yp telomeric (green) probes (16 h hybridization). (C) 1q telomeric (red) probe (16 h hybridization). (D) Y satellite (green) and 1 satellite (red) probes (3 h hybridization). (E) Y satellite (blue), Xp/Yp telomeric (green) and Xq/Yq telomeric (red) probes (16 h hybridization). (F) 1 centromeric (red) probe (3 h hybridization). DNA is stained blue with DAPI. Der(1) has an intact 1p short arm and a positive signal for the 1 centromere, followed by the translocated distal Yq12 heterochromatin and the Yq telomere. Der(Y) shows positive signals for the Yp telomere, Yp SRY, Y centromere and Yq12 proximal heterochromatin, followed by the 1q long arm containing the 1q telomere but not the 1q heterochromatin, which was lost in the translocation.

 
The karyotype was then interpreted as (Mitelman, 1995Go): 46,X,t(Y;1)(q12;q12).ish t(Y;1)(DXYS129+,SRY+,DYZ3+,DYZ1+,Z43206-,D1S3739+;D1Z3+,D1Z1–,DYZ1+,Z43206+).

Molecular studies confirmed the intactness of the SRY in Yp and of the Yq11.2 euchromatic region as shown by the absence of microdeletions in regions AZFa, AZFb and AZFc (Figure 3A). The study of DAZ gene copies revealed a polymorphic event at the proximal part of DAZ2 (del DAZ2p). This was shown by the presence of alleles A and B for all SNVs but only the B allele in SNV III, and the presence of RRM3 (DAZ1 and DAZ4), DAZ3 (DAZ3) and sY152 (DAZ1 and DAZ4) (Figure 3B). The intactness of the distal heterochromatic region of the Y chromosome was demonstrated by the positive reactions of STSs (sY160, sY1124, sY1245) for Yq12 (Figure 3C). It was not possible to check for genes located within the pseudoautosomal regions (PARs) because the STSs described presented high homologies with the same regions of the X chromosome.



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Figure 3. (A) Agarose gel electrophoresis of multiplex-PCR showing integrity of Yp (SRY) and absence of Yq11.2 microdeletions in regions AZFa, AZFb and AZFc. mw=molecular weight markers; P=patient; Mf=control fertile male; F=control female. (B)) DAZ single nucleotide variants (SNV I–VI) and STS markers (Triplex) used to analyse deletions in the four DAZ genes. Allele A was defined as not restricted and allele B as restricted (two fragments), whereas allele C in SNV V is a constant finding. The absence of allele A in SNV III defines the presence of a polymorphic deletion of proximal DAZ2. (C) The integrity of the proximal Yq12 heterochromatin region is shown by the positive amplification of three STSs.

 
Analysis of the fresh testicular seminiferous tissue revealed only a few germ cells whose morphology was compatible with arrest at meiosis I (Figure 4A). Some of these formed giant multinucleated cells (Figure 4B) that degenerated by vacuolization (Figure 4C), whereas most of the others were in the process of phagocytosis by Sertoli cells (Figure 4D and E). Interphase FISH analysis of isolated germ cells arrested at meiosis I revealed a significant decrease (P=0.005) of the frequency of sex chromosome pairing by PAR1 of Xp/Yp, which was found in only 19 out of 102 (18.6%) of the cells, in relation to the frequency of sex chromosome pairing found in controls (71 out of 108, 65.7%). Autosome pairing was found in all cases, being complete in 102 out of 125 (81.6%) and partial in 13 out of 125 (10.4%) of the cells (Figure 5). Long-term in vitro co-cultures of germ cells with Sertoli cells, under FSH and testosterone supplementation, were unable to rescue the meiotic arrest, with absence of germ cell cluster formation, either alone or associated with Sertoli cells, and with total degeneration of germ cells after the 2 week culture period (Figure 4F). mRNA expression studies confirmed that germ cells degenerated by apoptosis. This included the activation of several proapoptotic mechanisms, including the Fas ligand/Fas receptor and caspase-8 exogenous initiator pathway, the Bax and caspase-9 mitochondrial initiator pathway and the caspase-3 executioner pathway (Figure 6).



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Figure 4. Morphology of testicular germ cells. (A) Primary spermatocytes. (B) Giant intact multinucleated (n) primary spermatocytes. (C) Vacuolar degeneration (*) of giant multinucleated primary spermatocytes. (D and E) Sertoli cells (S) with phagocytosed germ cells (*). (F) Only Sertoli cells appear intact after a 2 week in vitro co-culture. Hoffman inverted microscopy.

 


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Figure 5. Sequential interphase FISH in testicular germ cells. (A and A') Xp/Yp pairing and partial autosome pairing (18–18 paired; 7–7 partial pairing). (B and B') Xp/Yp unpairing and complete autosome pairing. Y satellite (violet), Y centromeric (red), X centromeric (yellow) and Xp/Yp telomeric (green) probes (16 h hybridization); and 18 centromeric (green) and 7 centromeric (red) probes (3 h hybridization).

 


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Figure 6. Agarose gel electrophoresis of apoptotic gene mRNA expression. wm = molecular weight markers; Lr = lymphocytes from a leukaemic patient at remission; W1 = patient fresh testicular tissue, with intact Sertoli cells and degenerating germ cells; W2 = patient testicular tissue after a 2 week in vitro co-culture, with intact Sertoli cells and absence of germ cells. {beta}2-microglobulin was used as the internal control for caspases (casp) 9, 8 and 3, and glucose-6-phosphate dehydrogenase (G6PDH) for Fas receptor, Bax and Bcl2.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
To our knowledge, there are only three reports describing five cases with a balanced reciprocal (Y;1) translocation, all associated with male infertility but none involving the q12 bands of both chromosomes (Hsu, 1994Go; Maraschio et al., 1994Go; Pabst et al., 2002Go). A reciprocal (Y;1) translocation with breakpoints at q12 of both chromosomes but without loss of the heterochromatic region of chromosome 1 has only been reported in patients with malignant haematological disorders (Michaux et al., 1996Go). We here report a de novo balanced reciprocal t(Y;1)(q12;q12) translocation with breakpoints at Yq12 and 1q12 and loss of the heterochromatic region of chromosome 1, in a male with an otherwise normal phenotype besides azoospermia due to germ cell meiotic I arrest.

During the male meiotic prophase I, pairing between the X and Y chromosomes occurs in primary spermatocytes at the zygotene and pachytene stages, forming a condensation called the sex vesicle. Sex chromosome pairing appears distinct from autosome pairing, as it is limited to the telomere PARs, PAR1 (at Xp/Yp) and PAR2 (at Xq/Yq). Pairing between sex chromosomes begins in Xpter and Ypter, with synapses occurring in most parts of Yp and in the distal third of Xp. Deletions of pseudoautosomal sequences cause a lack of pairing between the X and Y chromosomes, thus indicating that there is an obligatory recombination event in the DNA homologue segment located in Xpter and Ypter during meiosis I, which is essential to promote meiotic pairing and ensure sperm production (Mohandas et al., 1992Go). Translocations involving a sex chromosome and an autosome are more prone to cause infertility than translocations involving autosomes. This is explained by the fact that the derivative chromosomes will interfere with normal sex and autosome homologue pairing and thus inhibit homologue segregation (Dutrillaux and Gueguen, 1975Go; Nielsen and Rasmussen, 1976Go; Mattei et al., 1978Go; Pajares et al., 1979Go; Smith et al., 1979Go; Gonzalez et al., 1981Go; Disteche et al., 1986Go; Moreau et al., 1987Go; Arnemann et al., 1991Go; Teyssier et al., 1993Go; Gardner and Sutherland, 1996Go; Delobel et al., 1998Go; Giltay et al., 1999Go; Powell, 1999Go).

In the present case, the breakpoint in the Y chromosome occurred in the distal Yq12 heterochromatic region. However, there was an intact SRY in Yp, no microdeletions in the AZFa, AZFb and AZFc regions, and the presence of markers for the proximal Yq12 heterochromatin. Gene copy-specific deletion analysis of the four DAZ gene copies revealed a deletion of the proximal part of DAZ2. However, deletion of DAZ2p has been shown to be a polymorphic event not related to oligozoospermia (Fernandes et al., 2002Go, 2004Go) or azoospermia (Ferrás et al., 2004Go). The intactness of Yp, Yq11.2 and proximal Yq12 thus discards the hypothesis that spermatogenesis failure could be due to the deletion of loci controlling germ cell differentiation. On the contrary, although metaphase FISH showed that the PAR1 region in Yp was not affected, allowing sex chromosome pairing initiation, the PAR2 region in Yq was translocated to der(1), which could interfere with sustained pairing, recombination and segregation. This was confirmed by interphase FISH in germ cells, which demonstrated that only 18.6% of primary spermatocytes exhibited sex chromosome pairing at PAR1. Furthermore, the large 1q segment translocated onto the distal Yq could also disturb X–der(Y) and der(1)–1 pairing, recombination and normal segregation. Therefore, the present data strongly indicate that azoospermia due to meiosis I arrest was effectively caused by the translocation.

Germ cell arrest has been suggested to induce degeneration of most spermatocytes by apoptosis, thus leading to a progressive loss of germ cells in the seminiferous tubules (Delobel et al., 1998Go). This is first demonstrated here by the fact that germ cells form giant multinucleated cells that subsequently exhibited vacuolar degeneration, the intense phagocytosis of germ cells by Sertoli cells and the activation of proapoptotic gene expression through exogenous (FasR/caspase-8/caspase-3) and endogenous (Bax/caspase-9/caspase-3) pathways.

In conclusion, we describe a de novo balanced reciprocal t(Y;1)(q12;q12) translocation with breakpoints at Yq12 and 1q12 and loss of the heterochromatic region of chromosome 1, in a male with an otherwise normal phenotype besides presenting azoospermia. The presence of an intact PAR1 region, the absence of Y chromosome microdeletions in the AZF and DAZ regions, the presence of proximal Yq12 heterochromatin markers, the presence of the Y-PAR2 region in der(1) and the loss of sex chromosome pairing at meiosis I indicate that arrest of spermatogenesis at zygotene/pachytene was caused by the translocation. We could also determine that pre-meiotic germ cell loss occurs continuously in the seminiferous tubules by Fas receptor-induced apoptosis with phagocytosis by Sertoli cells.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was partially supported by FCT (36363/99, 43462/01; 35231/99, 42812/01, 48376/02; UMIB).


    References
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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 20, 2004; resubmitted on July 9, 2004; accepted on November 10, 2004.





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