1 Department of Medical Genetics and 2 Reproductive Medicine Unit, I.R.C.C.S. `Saverio de Bellis`, 70013 Castellana Grotte (BA), Italy
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Abstract |
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Key words: alternate centromere inactivation/male infertility/Y-autosome translocation
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Introduction |
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Besides sex chromosome numerical aberrations, several structural abnormalities such as translocations, markers or inversions are more frequently found in the karyotype of infertile men (Thielemans et al., 1998; Gekas et al., 2001
). Numerous translocations have been associated with impaired spermatogenesis (Chandley, 1988
). An extremely rare aberration is the translocation of Y chromosomal DNA to an autosome, resulting in a phenotypically normal male with an unbalanced karyotype constituted by 45 chromosomes, including a dicentric chromosome deriving from the Y+autosome fusion.
Different hypotheses have been made as to correlations between Y/A translocations and spermatogenetic disturbances. The long (q) arm of the Y chromosome (Yq11) contains various genes required for normal spermatogenesis (AZFs, AZoospermia Factors) (Tiepolo et al., 1976; Pryor et al., 1997
; Liow et al., 2001
). On these bases, some authors (Vogt et al., 1995
, 1996
) have postulated the relevance of the Yq breakpoint in Y-autosome translocations, assuming the oligo/azoospermia to be strictly related to partial or complete loss of the AZF loci within the translocation-derived acentric fragment.
Other evidence (Smith et al., 1979; Laurent et al., 1982
; Delobel et al., 1998
) implies that oligo/azoospermia is the result of an abnormal sex vesicle formation with meiotic disturbances and consequent spermatogenetic arrest. In fact, spermatogenesis seems more vulnerable than oogenesis to chromosomal rearrangements. In particular, the involvement of an acrocentric chromosome negatively affects the mechanics of meiosis, predisposing to male infertility (Guichaoua et al., 1990
).
Here we describe a patient investigated for azoospermia, presenting a t(Y;14) with alternate centromeric inactivation on the peripheral blood karyotype. Cytogenetic, fluorescent in-situ hybridization (FISH) and molecular studies have been performed to gain a better understanding of the correlation between Y-autosome translocations and male infertility status.
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Materials and methods |
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Cytogenetic and FISH studies
Chromosome analysis was performed according to standard methods on cultured cells from the patient's peripheral blood. High resolution prometaphase chromosomes were examined by trypsin G (GTG) banding (Figure 1), quinacrine fluorescent (QFQ) banding, and 4'-6'-diamidino-2'-phenylindole/distamycin A (DAPI/DA) staining.
FISH analysis was performed as previously described (Gentile et al., 1993), using the following probes: chromosome 14/22 (D14Z1/D22Z1) and chromosome Y (DYZ1) centromeric specific probes; a yeast artificial chromosome (YAC) contig spanning the entire euchromatic region of the Yq (Figure 2
) (courtesy Dr M.Rocchi).
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Molecular analysis
The Yq chromosomal breakpoint was more precisely assessed by polymerase chain reaction analysis (PCR). Genomic DNA was prepared from peripheral blood lymphocytes (Nucleon BACC3; Amersham Pharmacia Biotech, Bucks, UK) and amplified in multiplex PCR containing two to six primer pairs. Two primers were amplified in a single reaction. The reaction products were analysed on 3% agarose gel (Metaphor, FMC, Rockland, ME, USA) and visualized with ethidium bromide. The patient was screened for 27 sequence-tagged-sites (STS) specific to the different Y chromosome AZF loci (Figure 3) (Reijo et al., 1996
; Stuppia et al., 1996
; Kent-First et al., 1999
).
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Results |
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Two-colour FISH analysis with Y and 14/22 alpha-satellite centromeric specific probes confirmed the presence of both centromeres (Figure 2A). Hybridization with six YAC clones, covering the entire Yq euchromatic region, showed no deletion, allowing correct breakpoint location at Yq12, with loss restricted to the Yq heterochromatin (Figure 2B
, C, D).
The karyotype was 45,X,dic(Y;14)(q12;p11) de novo.ish (D14Z1/D22Z1+; DYZ1+).
Molecular analysis
DNA analysis showed amplification products in all 27 STS analysed, indicating that the patient carried the Yp, the centromere, and the euchromatic portion of the long arm of the Y chromosome. The distal location of the sY160 marker at Yq12 confirmed that the Y chromosome interval 7 was retained, as well as the breakpoint assignation to the junction between the euchromatic and heterochromatic segments of the Y chromosome.
Sex chromosomes specific amplifications on eight spermatids revealed the presence of both signals in five cases, the Y chromosome signal in two, and no signals in one (Figure 3).
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Discussion |
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Y-autosome translocations can be subdivided into different groups according to the Y chromosome breakpoints and/or autosome involved. In the most common form, the autosome is an acrocentric with the breakpoint in the short arm and the Y breakpoint at the q arm heterochromatin junction. Usually these translocations are balanced, with the presence of one derivative chromosome looking rather a sub-metacentric with a short arm constituted by the Q fluorescent Yq heterochromatin (Yqh) (Cohen et al., 1981). These cases, frequently familial, do not affect fertility and can be regarded as of no clinical significance (Chandley, 1988
).
Very rarely, the same translocation can result in an unbalanced karyotype with 45 chromosomes. In these cases the Yq and acrocentric p breakpoints fuse differently, resulting in a dicentric chromosome containing the Yp, the centromere, the Yq euchromatic region, the acrocentric centromere and long arm. Although these patients are phenotypically males, a major concern is the effect of these aberrations on their fertility, as azoospermia/hypogonadism is a very frequent feature (Chandley, 1988). Different hypotheses have been advanced to explain such associations.
In the last 10 years, numerous studies have demonstrated Yq euchromatin microdeletions in men with severe impairment of spermatogenesis (AZF loci) (Pryor et al., 1997; Maurer and Simoni, 2000
; Liow et al., 2001
). In this view, male infertility could represent the result of translocation breakpoint rearrangements with loss of Yq loci involved in spermatogenesis distally to the Y breakpoint (Erickson et al., 1995
). The absence of detailed Y breakpoint characterization in previously reported cases (Smith et al., 1979
; Laurent et al., 1982
; Viguié et al., 1982
; Callen et al., 1987
; Moreau et al., 1987
; Matsuda et al., 1989
; Abbas et al., 1990
; Teyssier et al., 1993
; Farah et al., 1994
; Giltay et al., 1998
) makes it difficult to verify this hypothesis. However in our case, as in one previously described (Delobel et al., 1998
), this kind of mechanism is quite improbable: FISH and molecular studies clearly demonstrate retention of the entire Yq euchromatic region and AZF loci.
A well known phenomenon reported in literature is the relation between meiotic disturbances and spermatogenetic arrest (Guichaoua et al., 1991; Maraschio et al., 1994
). Chromosomal rearrangements involving an autosome and a sex chromosome alter sex vesicle formation, with loss of the asynchronous control of the sex chromosome and autosomal gene transcription (Lifschytz and Lindsley, 1972
; Delobel et al., 1998
). In particular, in the presence of Y-autosome translocations, segments of the autosome might be included in the sex vesicle with consequent hypercondensation and inactivation resulting in a severe spermatogenetic disorder (Laurent et al., 1982
; Delobel et al., 1998
). A similar mechanism presumably occurs in X-autosome translocation, causing male infertility in almost all cases (Schmidt and Du Sart, 1992
).
The spermatogenic block mainly occurs at the first meiotic division (primary spermatocyte stage). Occasionally, some germ cells can escape this arrest and continue until the spermatid stage, as confirmed by the fortuitous recovery of elongated spermatids in our patient's ejaculate (Tesarik et al., 1998).
In addition, our patient's translocation showed an alternate centromere inactivation, a very unusual cytogenetic phenomenon, rarely described in patients with heterodicentric chromosomes (Ing et al., 1983; Rivera et al., 1989
; Fisher et al., 1997
). Different hypotheses have been put forward as to the significance and the genetic mechanisms associated with centromeric inactivation. Fisher et al. suggest that loss of the kinetochore has a relevant role in inducing the inactive state (Fisher et al., 1997
). The identical composition of the mitotic kinetochore in neocentromeres and normal centromeres, as well as the complete absence of kinetochore from inactive centromeres found on dicentric chromosomes, suggest that the alpha DNA may be essential, but not sufficient for centromeric activity, underlining the relevance of epigenetic mechanisms in human centromere formation (Tyler-Smith et al., 1998
; Warburton, 2001
). Nevertheless, the reasons for the alternate centromeric inactivation seen in some dicentric chromosomes and the possible phenotypic and clinical relevance of this event are poorly understood. The finding of both 14 and 21 active forms in our case suggests that the two centromeres were initially active. In addition, similarly to the case described by Ing et al., preferentially acrocentric (14 chromosome) centromere activation is present (Ing et al., 1983
). No relation can be established between alternate inactivation and spermatogenetic defects. However, the presence of this pattern further underlines the chromosomal complexity of our case and the potential role of meiotic mechanism disruption in spermatogenetic arrest.
A last, important, question regards the correct estimate of the level of risk of the couple having chromosomally abnormal embryos and an abnormal/infertile child. In fact, in most cases Y-autosome carriers are infertile, but the recent advances in ICSI bypass this status even in the presence of severely impaired semen parameters. To assess the genetic risk, we collected eight elongated spermatids and analysed them for sex chromosomes. Our data, although restricted in number, seem to indicate the prevalence of cells containing the Y;14 and X chromosomes (5/8) (Figure 3), with a theoretically increased risk of having chromosomally unbalanced offspring.
A similar study has been performed in a patient with a t(Y;16)(q11.21;q24) (Giltay et al., 1999). The authors examined sperm, detecting 49% of unbalanced sperm cells; the percentage increased to nearly 90% when morphologically abnormal cells were included. Despite some differences in the translocation (autosome involved; Yq breakpoint), the technique (FISH), and, above all, the seminal parameters (presence of sperm), these studies support the presence of an increased risk and can be seen as grounds for offering preimplantation and/or prenatal diagnosis in such cases.
In conclusion, the cytogenetic and molecular studies of this case suggest that the correlation between Y-autosome translocations and male infertility can be explained in terms of meiotic mechanism vulnerability by unpaired autosomal segments, particularly of acrocentric chromosomes. Other factors such as the autosome involved and/or the genetic background could contribute to determine the semen fertility potential (Delobel et al., 1998). Preliminary data indicate a high prevalence of chromosomally unbalanced sperm cells, stressing the potential transmission of the chromosomal abnormality to progeny when fertilization is assisted by appropriate micromanipulation techniques.
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Acknowledgements |
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Notes |
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References |
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Callen, D.F., Sutherland, G.R. and Carter, R.F. (1987) A fertile man with tdic(Y;22): how a stable neo-X1X2Y sex determining mechanism could evolve in man. Am. J. Med. Genet., 3, 151155.
Chandley, A.C. (1988) Meiotic studies and fertility in human translocation carriers. In Daniel, A. (ed.), The Cytogenetics of Mammalian Autosomal Rearrangements. A.R.Liss, New York, pp. 361382.
Chandley, A.C. and Haergreave, T.B. (1996) Genetic anomaly and ICSI. Hum. Genet., 11, 930932.
Cohen, M.M., Frederick, R.W., Balkin, N.E. and Simpson, N.J. (1981) The identification of Y chromosome translocations following dystamicin A treatment. Clin. Genet., 19, 335342.[ISI][Medline]
Delobel, B., Djlelati, R., Gabriel-Robez, O., Croquette, M.F. et al. (1998) Y-autosome translocation and infertility: usefulness of molecular, cytogenetic and meiotic studies. Hum. Genet., 102, 98102.[ISI][Medline]
Erickson, R.P., Hudgins, L., Stone, J.F. et al. (1995) A `balanced' Y;16 translocation associated with Turner-like neonatal lymphedema suggests the location of a potential anti-Turner gene on the Y chromosome. Cytogenet. Cell, Genet., 71, 163167.[ISI][Medline]
Farah, S.B., Ramos, C.F., de Mello, M.P. et al. (1994) Two cases of Y;autosome translocations: a 45,X male and a clinically trisomy 18 patient. Am. J. Med. Genet., 49, 388392.[ISI][Medline]
Fisher, A.M., Al-Gazali, L., Pramathan, T. et al. (1997) Centromeric inactivation in a dicentric human Y;21 translocation chromosome. Chromosoma, 106, 199206.[ISI][Medline]
Fonatsch, C. and Streubel, B. (1998) Classical and molecular cytogenetics. In Huhn, D. (ed.), New Diagnostic Methods in Oncology + Hematology. Springer-Verlag, Berlin.
Gekas, J., Thepot, F., Turleau, C. et al. (2001) Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Hum. Reprod., 16, 8290.
Gentile, M., Susca, F., Resta, N. et al. (1993) Infertility in carriers of two bisatellited marker chromosomes. Clin. Genet., 44, 7175.[ISI][Medline]
Giltay, J.C., Tiemessen, C.H., Van Inzen, W.G. and Scheres, J.M. (1998) One normal child and a chromosomally balanced/normal twin after intracytoplasmic sperm injection in a male with a de-novo t(Y;16) translocation. Hum. Reprod., 13, 27452747.
Giltay, J.C., Kastrop, P.M., Tiemessen, C.H. et al. (1999) Sperm analysis in a subfertile male with a Y;16 translocation, using four-color FISH. Cytogenet. Cell, Genet., 84, 6772.[ISI][Medline]
Guichaoua, M.R., Quack, B., Speed, M.R. et al. (1990) Infertilty in human males with autosomal translocations: meiotic study of a 14;22 Robertsonian translocation. Hum. Genet., 86, 162166.[ISI][Medline]
Guichaoua, M.R., de Lanversin, A., Cataldo, C. et al. (1991) Three dimensional reconstruction of human pachytene spermatocyte nuclei of a 17;21 reciprocal translocation carrier: study of XY-autosome relationship. Hum. Genet., 87, 709715.[ISI][Medline]
Haergreave T. (2000) Genetically determined male infertility and assisted reproduction techniques. J. Endocrinol. Invest., 23, 697710.[ISI][Medline]
Ing, S.P. and Smith, S.D. (1983) Cytogenetic studies of a patient with mosaicism of isochromosome 13q and a dicentric (Y;13) translocation showing differential centromeric activity. Clin. Genet., 24, 194199.[ISI][Medline]
Kent-First, M., Muallem, A., Shultz, J. et al. (1999) Defining regions of the Y-chromosome responsible for male infertilty and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol. Reprod. Dev., 53, 2741.[ISI][Medline]
Laurent, C., Chandley, A.C., Dutrillaux, B. and Speed, R.M. (1982) The use of surface spreading in the pachytene analysis of a human t(Y;17) reciprocal translocation. Cytogenet. Cell. Genet., 33, 312318.[ISI][Medline]
Lifschytz, E. and Lindsley, D.L. (1972) The role of X-chromosome inactivation during spermatogenesis. Proc. Natl Acad. Sci. USA, 69, 182186.[Abstract]
Liow, S.L., Yong, E.L. and Ng, S.C. (2001) Prognostic value of Y deletion analysis. Hum. Reprod., 16, 912.
Maraschio, P., Tupler, R., Dainotti, E. et al.(1994) Molecular analysis of a human Y;1 translocation in an azoospermic male. Cytogenet. Cell. Genet., 65, 256260.[ISI][Medline]
Matsuda, T., Hayashi, K., Nonomura, M. et al. (1989) Azoospermic male with a balanced Y-autosome translocation. Urol. Int., 44, 4346.[ISI][Medline]
Maurer, B. and Simoni, M. (2000) Y chromosome microdeletion screening in infertile men. J. Endocrinol. Invest., 23, 664670.[ISI][Medline]
Moreau, N., Teysser, 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]
Pryor, J.L., Kent-First, M., Muallem, A. et al. (1997) Microdeletions in the Y chromosome of infertile men. N. Engl. J. Med., 336, 534539.
Reijo, R., Alagappan, R.K., Patrizio, P. and Page, D.C. (1996) Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet, 347, 12901293.[ISI][Medline]
Rivera, H., Zuffardi, O., Maraschio, P. et al. (1989) Alternate centromere inactivation in a pseudodicentric (15;20)(pter;pter) associated with a progressive neurological disorder. J. Med. Genet., 26, 626630.[Abstract]
Sasabe, Y., Stehlik, E.F., Krisher, R.L. et al. (1996) Sex determination by simultaneous application of polymerase chain reaction and fluorescent in situ hybridization on the same blastomere of a pre-embryo. Fertil. Steril., 66, 490492.[ISI][Medline]
Schmidt, M. and Du Sart, D. (1992) Functional disomies of the X chromosome influence the cell selection and hence the X inactivation pattern in females with balanced X-autosome translocations: a review of 122 cases. Am. J. Med. Genet., 42, 161169.[ISI][Medline]
Smith, A., Fraser, I.S. and Elliot, G. (1979) An infertile male with balanced Y;19 translocation. Review of Y;autosome translocations. Ann. Genet., 22, 189194.[ISI][Medline]
Stuppia, L., Gatta, V., Calabrese, G. et al. (1996) A quarter of men with idiopathic oligo-azoospermia display chromosomal abnormalities and microdeletions of different types in interval 6 of Yq11. Hum. Genet., 102, 566570.
Tesarik, J., Sousa, M., Greco, E. and Mendoza, C. (1998) Spermatids as gametes: indications and limitations. Hum. Reprod., 13, 89105.[Medline]
Teyssier, M., Rafat, A. and Pugeat, M. (1993) Case of (Y;1) familial translocation. Am. J. Med. Genet., 46, 339340.[ISI][Medline]
Thielemans, B.F.J., Spiessens, C., D'Hooghe, T. et al. (1998) Genetic abnormalities and male infertility. A comprehensive review. Eur. J. Obstet. Gynecol., 81, 217225.[ISI][Medline]
Tiepolo, L. and Zuffardi, O. (1976) Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum. Genet., 34, 119124.[ISI][Medline]
Tyler-Smith, C., Corish, P. and Burns, E. (1998) Neocentromeres, the Y chromosome and centromere evolution. Chrom. Res., 6, 6567.[Medline]
Viguié, F., Romani, F. and Dadoune, J.R. (1982) Male infertility in a case of (Y;6) balanced reciprocal translocation. Hum. Genet., 62, 225227.[ISI][Medline]
Vogt, P.H., Edelmann, A., Hirschmann, P. and Kohler, M.R. (1995) The azoospermia factor (AZF) of the human Y chromosome in Yq11: function and analysis in spermatogenesis. Reprod. Fertil. Dev., 7, 685693.[ISI][Medline]
Vogt, P.H., Edelmann, A., Kirsch, S. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet., 5, 933943.
Warburton, P.E. (2001) Epigenetic analysis of kinetochore assembly on variant human centromeres. Trends Genet., 17, 243246.[ISI][Medline]
accepted on November 2, 2001.