Molecular and cytogenetic characterization of an azoospermic male with a de-novo Y;14 translocation and alternate centromere inactivation

A.L. Buonadonna1, F. Cariola1, E. Caroppo2, A.Di Carlo1, P. Fiorente1, M.C. Valenzano1, G. D'Amato2 and M. Gentile1,3

1 Department of Medical Genetics and 2 Reproductive Medicine Unit, I.R.C.C.S. `Saverio de Bellis`, 70013 Castellana Grotte (BA), Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Y-autosome (Y/A) translocations have been reported in association with male infertility. Different hypotheses have been made as to correlations between Y/A translocations and spermatogenetic disturbances. We describe an azoospermic patient with a de-novo Y;14 translocation: 45,X,dic(Y;14)(q12;p11). METHODS AND RESULTS: Cytogenetic, fluorescent in-situ hybridization (FISH) and molecular studies have been performed. A 14/22 (D14Z1/D22Z1) centromere and a Y centromere (DYZ1) probe both showed a signal on the translocation chromosome, confirming its dicentricity. Each copy of the translocation chromosome had only one primary constriction, with inactivation of the Y centromere in most (90%) of the cells. The 14 centromere was inactive in the remaining cells (10%). FISH and molecular deletion mapping analysis allowed acute assignment of the Yq breakpoint to the junction of euchromatin and heterochromatin (Yq12), distal to the AZF gene location (Yq11). CONCLUSIONS: This study supports the hypothesis that in Y/A translocations infertility might be related to meiotic disturbances with spermatogenetic arrest. In addition, sex chromosome molecular investigations, performed on single spermatids, suggest a highly increased risk of producing chromosomally abnormal embryos.

Key words: alternate centromere inactivation/male infertility/Y-autosome translocation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The widespread use of ICSI technology has significantly improved the outlook for couples with severe male infertility (Haergreave et al., 2000Go). Genetic abnormalities account for a large proportion of male infertility, and consequently, by avoiding all the steps of natural selection, ICSI permits the transmission of genetically determined defects of spermatogenesis or other genetic defects to male offspring (Chandley and Haergreave, 1996Go). In particular, a constitutional chromosomal abnormality is present in 2.2–8.6% of infertile men, increasing to 16% in azoospermic patients (Thielemans et al., 1998Go; Gekas et al., 2001Go).

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., 1998Go; Gekas et al., 2001Go). Numerous translocations have been associated with impaired spermatogenesis (Chandley, 1988Go). 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., 1976Go; Pryor et al., 1997Go; Liow et al., 2001Go). On these bases, some authors (Vogt et al., 1995Go, 1996Go) 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., 1979Go; Laurent et al., 1982Go; Delobel et al., 1998Go) 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., 1990Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clinical report
A 29-year-old man and a 30-year-old woman were referred to our centre because of infertility. No family history of congenital anomalies, hereditary diseases, or infertility was present in the couple. Both were normal at physical examination, including their reproductive systems. Semen analyses showed azoospermia. Spermatocytes and rare elongated spermatids were observed. At histological examination, thick membranes of the seminiferous tubules were seen. A spermatogenic hypoplasia was present, with a severely reduced population of germ cells in different stages, and a poor order of spermatogenesis. Very few spermatids, but no mature sperm, were present in the seminiferous tubules lumen (Figure 1Go).



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Figure 1. . (A) Testis biopsy histology showing disorganization of the seminiferous tubules, with clusters of spermatogenic cells in different degrees of development. Very few spermatids are present in the lumen (B). (C, D) Partial karyotype showing GTG banded chromosomes. The arrows indicate the 14 chromosome (arrow head) and the t(Y;14) dicentric chromosome. (C) In most of the cells (90%) the Y chromosome was inactivated (grey arrow), and the chromosome 14 centromere active (black arrow). (D) In the other cells (10%) the 14 centromere was inactive (grey arrow), and the Y centromere active (black arrow).

 
FSH, LH, estradiol, testosterone and prolactin concentrations were within normal ranges.

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 1Go), 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., 1993Go), 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 2Go) (courtesy Dr M.Rocchi).



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Figure 2. . FISH analysis showing two signals for the 14 and Y alpha-satellite centromeric probes (A); YAC contig two-colour hybridization signals (B, C, D) with YAC clones locations on the Y chromosome ideogram.

 
Preparation and labelling of YAC clones and two-colour FISH were performed according to current methods (Fonatsch and Streubel, 1998Go). Biotin and digoxygenin-labelled probes were detected with rhodamine or fluorescein isothiocyanate (FITC) and chromosomes were counterstained with DAPI. Images were captured with a PSI (Perceptive Scientific Image, Chester, UK) system.

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 3Go) (Reijo et al., 1996Go; Stuppia et al., 1996Go; Kent-First et al., 1999Go).



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Figure 3. . Agarose gel electrophoresis of PCR products of: (A) Y chromosome STS with ideogram showing Y chromosome mapping;(B) single spermatids showing the 130 bp (X) and 154 bp (Y) bands in five cells (1, 3, 6, 7, 8); the 154 bp band in two (4, 5), and no bands in one (2). S, size markers; M, male; F, female; B, blank.

 
Eight elongated spermatids were selected, aspirated into the injection pipette, transferred to a 0.5 ml microcentrifuge tube with 100 ml PCR reaction mixture and amplified with Y and X-chromosome specific primers according to a previously described method (Sasabe et al., 1996Go). The amplification products were visualized with ethidium bromide on a 3% agarose gel (Figure 3Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cytogenetic and FISH
High resolution karyotyping in our patient showed 45 chromosomes in each of 100 cells, with a dicentric (Y;14) translocation apparent in two configurations. In fact, in most (90%) of the cells, the Y chromosome centromere was inactivated and the chromosome 14 centromere constricted (Figure 1CGo). In the remaining cells (10%), the 14 chromosome was inactivated (Figure 1DGo). No Q and DA/DAPI heterochromatic regions on Yq12 were present. Parental chromosomes were normal.

Two-colour FISH analysis with Y and 14/22 alpha-satellite centromeric specific probes confirmed the presence of both centromeres (Figure 2AGo). 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 2BGo, 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 3Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We describe a unique Y;14 translocation with alternate centromere inactivation in an azoospermic male. Cytogenetic, FISH, and molecular analyses have been made to allow accurate assignment of the breakpoint location, and, finally, to define genotype-phenotype correlations and potential genetic risk to offspring.

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., 1981Go). These cases, frequently familial, do not affect fertility and can be regarded as of no clinical significance (Chandley, 1988Go).

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, 1988Go). 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., 1997Go; Maurer and Simoni, 2000Go; Liow et al., 2001Go). 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., 1995Go). The absence of detailed Y breakpoint characterization in previously reported cases (Smith et al., 1979Go; Laurent et al., 1982Go; Viguié et al., 1982Go; Callen et al., 1987Go; Moreau et al., 1987Go; Matsuda et al., 1989Go; Abbas et al., 1990Go; Teyssier et al., 1993Go; Farah et al., 1994Go; Giltay et al., 1998Go) makes it difficult to verify this hypothesis. However in our case, as in one previously described (Delobel et al., 1998Go), 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., 1991Go; Maraschio et al., 1994Go). 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, 1972Go; Delobel et al., 1998Go). 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., 1982Go; Delobel et al., 1998Go). A similar mechanism presumably occurs in X-autosome translocation, causing male infertility in almost all cases (Schmidt and Du Sart, 1992Go).

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., 1998Go).

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., 1983Go; Rivera et al., 1989Go; Fisher et al., 1997Go). 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., 1997Go). 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., 1998Go; Warburton, 2001Go). 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., 1983Go). 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 3Go), 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., 1999Go). 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., 1998Go). 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Mr Pasquale Nanna and Mrs Nicoletta Tutino for technical help and Mary V.C.Pragnell for careful linguistic text revision. This study was supported by the following grants: Project 16/2001 IRCCS `S. de Bellis'.


    Notes
 
3 To whom correspondence should be addressed at: Department of Medical Genetics, I.R.C.C.S. `Saverio de Bellis`, via della Resistenza, 70013 Castellana Grotte (BA), Italy. E-mail: mattiagentile{at}libero.it Back

Submitted July 12, 2001


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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accepted on November 2, 2001.