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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: apoptosis/AZF/DAZ/meiosis I arrest/t(Y;1)(q12;q12) de novo balanced reciprocal translocation/Y chromosome
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1994; Maraschio et al., 1994
; Pabst et al., 2002
). 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., 1996
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1995). Fluorescence in situ hybridization (FISH) was performed as previously described (Alves et al., 2002
), using
-satellite probes for the centromeric regions of chromosomes X (DXZ1, Xp11.1q11.1; Vysis Inc., Downers Grove, IL), Y (DYZ3, Yp11.1-q11.1; Vysis) and 1 (D1Z5, 1p11.1q11.1; 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 (810 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., 2002).
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 IVI) and two STSs (DAZ-RRM3, Y-DAZ3) as previously described (Fernandes et al., 2002; Ferrás et al., 2004
). 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 TrisHCl, 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., 2002b). 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 1520 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., 1999
, 2001
; Sousa et al., 2002a
).
Meiotic studies
Sequential FISH was performed (Sousa et al., 2002a) using
-satellite probes (Vysis) for the centromeric regions of chromosomes X (DXZ1, Xp11.1q11.1), Y (DYZ3, Yp11.1q11.1), 7 (D7Z1, p11.1q11.1) and 18 (D18Z1, p11.1q11.1), 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., 1994; Teitz et al., 2000
, 2002
), 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
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.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1992). 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, 1975
; Nielsen and Rasmussen, 1976
; Mattei et al., 1978
; Pajares et al., 1979
; Smith et al., 1979
; Gonzalez et al., 1981
; Disteche et al., 1986
; Moreau et al., 1987
; Arnemann et al., 1991
; Teyssier et al., 1993
; Gardner and Sutherland, 1996
; Delobel et al., 1998
; Giltay et al., 1999
; Powell, 1999
).
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., 2002, 2004
) or azoospermia (Ferrás et al., 2004
). 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 Xder(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., 1998). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnemann J, Schnittger S, Hinkel GK, Tolkendorf E, Schmidtke J and Hansmann I (1991) A sterile male with 45,X0 and a Y;22 translocation. Hum Genet 87, 134138.[CrossRef][ISI][Medline]
Cremades N, Bernabeu R, Barros A and Sousa M (1999) In vitro maturation of round spermatids using coculture on Vero cells. Hum Reprod 14, 12871293.
Cremades C, Sousa M, Bernabeu R and Barros A (2001) Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids. Hum Reprod 16, 19381944.
Delobel B, Djlelati R, Gabriel-Robez O, Croquette M-F, Rousseaux-Prevost R, Rousseaux J, Rigot J-M and Rumpler Y (1998) Y-autosome translocation and infertility: usefulness of molecular, cytogenetic and meiotic studies. Hum Genet 102, 98102.[CrossRef][ISI][Medline]
Disteche CM, Brown L, Saal H, Friedman C, Thuline HC, Hoar DI, Pagon RA and Page DC (1986) Molecular detection of a translocation (Y;15) in a 45,X male. Hum Genet 74, 372377.[CrossRef][ISI][Medline]
Dutrillaux B and Gueguen J (1975) Etude méiotique et mitotique dans un cas de translocation t(5;Y). Humangenetik 27, 241245.[CrossRef][ISI][Medline]
Fernandes S, Huellen K, Gonçalves J, Dukal H, Zeisler J, de Meyts ER, Skakkebaek NE, Habermann B, Krause W, Sousa M, Barros A and Vogt PH (2002) High frequency of DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia. Mol Hum Reprod 8, 286298.
Fernandes S, Paracchini S, Meyer LH, Floridia G, Tyler-Smith C and Vogt PH (2004) A large AZFc deletion removes DAZ3/DAZ4 and nearby genes from men in Y haplogroup N. Am J Hum Genet 74, 180187.[CrossRef][ISI][Medline]
Fernandes-Alnemri T, Litwack G and Alnemri ES (1994) CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1-converting enzyme. J Biol Chem 269, 3076130764.
Ferrás C, Fernandes S, Marques CJ, Carvalho F, Alves C, Silva J, Sousa M and Barros A (2004) AZF and DAZ gene copy-specific deletion analysis in maturation arrest and Sertoli cell-only syndrome. Mol Hum Reprod 10, 755761.
Gardner RJM and Sutherland GR (1996) Chromosome Abnormalities and Genetic Counselling. In Sex Chromosome Translocations. Oxford University Press, Oxford, pp. 95114.
Giltay JC, Kastrop PMM, Tiemessen CHJ, Van Inzen WG, Scheres JMJC and Pearson PL (1999) Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH. Cytogenet Cell Genet 84, 6772.[CrossRef][ISI][Medline]
Gonzalez J, Lesourd S and Dutrillaux B (1981) Mitotic and meiotic analysis of a reciprocal translocation Y;13 in an azoospermic male. Hum Genet 57, 111114.[ISI][Medline]
Hsu LYF (1994) Phenotype/karyotype correlations of Y chromosome aneuploidy with emphasis on structural aberrations in postnatally diagnosed cases. Am J Med Genet 53, 108140.[ISI][Medline]
Mitelman F (1995) ISCN: An international system for human cytogenetic nomenclature. S Karger, Basel.
Maraschio P, Tupler R, Dainotti E, Cortinovis M and Tiepolo L (1994) Molecular analysis of a human Y;1 translocation in an azoospermic male. Cytogenet Cell Genet 65, 256260.[ISI][Medline]
Mattei JF, Mattei MG, Coignet J and Giraud F (1978) Y autosome translocation and complex chromosome rearrangement in cri du chat syndrome. J Med Genet 15, 154157.[Abstract]
Michaux L, Wlodarska I, Vellosa ER, Verhoef G, Van Orshoven A, Michaux JL, Scheiff JM, Mecucci C and Van den Berghe H (1996) Translocation (Y;1)(q12;q12) in hematologic malignancies. Report on two new cases, FISH characterization, and review of the literature. Cancer Genet Cytogenet 86, 3538.[CrossRef][ISI][Medline]
Mohandas TK, Speed RM, Passage MB, Yen PH, Chandley AC and Shapiro LJ (1992) Role of the pseudoautosomal region in sex-chromosome pairing during male meiosis: meiotic studies in a man with a deletion of distal Xp. Am J Hum Genet 51, 526533.[ISI][Medline]
Moreau N, Teyssier M and Rollet J (1987) A new case of (Y;1) balanced reciprocal translocation in an infertile man with Hodgkin's disease. J Med Genet 24, 379380.[ISI][Medline]
Morel F, Dugueperoux I, McElreavey K, le Bris M-J, Herry A, Parent P, le Martelot M-T, Fellous M and de Braekeleer M (2002) Transmission of an unbalanced (Y;1) translocation in Brittany, France. J Med Genet 39(e52), 15.
Nielsen J and Rasmussen K (1976) Y/autosomal translocations. Clin Genet 9, 609617.[ISI][Medline]
Pabst B, Glaubitz R, Schalk T, Schneider U, Schulze W and Miller K (2002) Reciprocal translocation between Y chromosome long arm euchromatin and the short arm of chromosome 1. Ann Génét 45, 58.[ISI][Medline]
Pajares IL, Delicado A, Cobos PV, Corral FS and Cuadrado C (1979) An azoospermic male with a Y/autosome translocation. Hum Genet 46, 155158.[CrossRef][ISI][Medline]
Powell C (1999) Sex chromosomes and sex abnormalities. In Gersen GL and Keagle MB (eds) The Principles of Clinical Cytogenetics. Humana Press, Totowa, NJ, pp. 229258.
Smith A, Fraser IS and Elliott G (1979) An infertile male with balanced Y;19 translocation. Review of Y;autosome translocations. Ann Génét 22, 189194.[ISI][Medline]
Sousa M, Cremades C, Alves C, Silva J and Barros A (2002a) Developmental potential of human spermatogenic cells cocultured with Sertoli cells. Hum Reprod 17, 161172.
Sousa M, Cremades N, Silva J, Oliveira C, Teixeira da Silva J, Viana P, Ferrás L and Barros A (2002b) Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozenthawed sperm and spermatids. Hum Reprod 17, 18001810.
Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, Behm FG, Look AT, Lahti JM and Kidd VJ (2000) Caspase-8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nat Med 6, 529535.[CrossRef][ISI][Medline]
Teitz T, Wei T, Liu D, Valentine V, Valentine M, Grenet J, Lahti JM and Kidd VJ (2002) Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN. Oncogene 21, 18481858.[CrossRef][ISI][Medline]
Teyssier M, Rafat A and Pugeat M (1993) Case of (Y;1) familial translocation. Am J Med Genet 46, 339340.[ISI][Medline]
Verma RS and Babu A (1995) Human Chromosomes. In Principles and Techniques. McGraw-Hill.
Vogt PH and Fernandes S (2003) Polymorphic DAZ gene family in polymorphic structure of AZF locus: artwork or functional for human spermatogenesis? APMIS 111, 115127.[CrossRef][ISI][Medline]
Submitted on May 20, 2004; resubmitted on July 9, 2004; accepted on November 10, 2004.
|