1 FECUNDITAS, Instituto de Medicina Reproductiva, Larrea 790, Buenos Aires and 2 Centro de Investigaciones en Reproducción (CIR), Facultad de Medicina, Paraguay 2155, Buenos Aires (1121), Argentina
3 To whom correspondence should be addressed. Email: asolari{at}fmed.uba.ar
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Abstract |
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Key words: azoospermia/complex chromosome rearrangement/FISH/human meiosis/synaptonemal complex
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Introduction |
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Most of the women with CCRs have been identified because they give birth to malformed children or have repeated abortions (Batista et al., 1994), while most of the males with CCRs were found in men showing infertility problems (Siffroi et al., 1997
). Usually, these men are sterile because of hypospermatogenesis or spermatogenic arrest. Presumably, this arrest stems from the complexity of the observed meiotic configurations (Chandley, 1981
).
CCRs were not described in any of the cytogenetic surveys of newborn infants (Jacobs et al., 1974; Hamerton et al., 1975
; Nielsen and Sillesen, 1975
). Therefore, the occurrence of CCRs is very low, and only
100 cases have been recorded (Joyce et al., 1999
; Cai et al., 2001
; Battisti et al., 2003
). In
70% of the cases, the patients had normal phenotypes and were detected because of reproductive problems.
The CCRs are classified into two groups: familial and de novo. These two groups are distinguished by the numbers of breakpoints, 4 in the former type and >4 in the latter (Kousseff et al., 1987
). In most of the familial cases (having between three and four breakpoints), the phenotype is normal in the apparently balanced carriers but they may have a significant risk of reproductive failure (Lespinasse et al., 2003
).
Half of the de novo CCRs have >4 breakpoints and are associated with multiple malformations, even though they are apparently balanced. Those with four breakpoints may show either abnormal or normal phenotype (Walker et al., 1985; Gorski et al., 1986
; Tupler et al., 1992
). As the CCRs are not always identified with conventional cytogenetic studies, fluorescence in situ hybridization (FISH) (Astbury et al., 2004
) and meiotic analysis (Solari and Rey Valzacchi, 1997
; Solari, 1999
) are useful tools for an accurate diagnosis.
We report here the case of an azoospermic male with a CCR, where three chromosomes are involved (12, 15 and Y) and four breakpoints were observed. The present study shows a deep disturbance of spermatogenesis occurring at the late pachytene stages which leads to the cell death of the vast majority of spermatocytes and the lack of spermiogenesis. The possible mechanisms underlying this disturbance are discussed.
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Materials and methods |
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Slide preparation
For chromosome analysis from peripheral blood cultures, slides were made following standard cytogenetic procedures (C-banding and G-banding). Fresh slides prepared in the same way were used for FISH. Microdeletions in the AZF region were tested with multiplex PCR for this segment of the Y chromosome.
FISH
FISH studies were performed according to the protocols provided by the manufacturer (Cambio Ltd, Cambridge, UK), using indirectly labelled, whole-chromosome painting (WCP) probes specific for the X, Y, 15 (Cambio Ltd) and Y centromeric, and a probe for the PraderWilliAngelman-specific locus (15q1113) (Oncor, Gaithersburg, MD). Another WCP probe was used for the entire chromosome 12 and a probe for chromosome 12pter was used to label this region. Both probes for chromosome 12 were obtained by microdissection in a local laboratory. Probe detection was carried out with fluorescein isothiocyanate (FITC; or rhodamine in the case of WCP for chromosome 12) and counterstained with 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). A Leica DM microscope fitted with a double-band pass filter and single-band filters was used for observation and registering of FITC and PI or DAPI in 35 mm negative film (400 ASA and 100 ASA, Kodak, Rochester, MN).
Testicular histology and fine structural observations of synaptonemal complexes
Bilateral testicular biopsies were performed as indicated by the andrologist after the approval of the corresponding ethics committee. Biopsies were indicated for routine histology and attempted sperm collection. The recovery of spermatozoa for ICSI treatment was tried with the largest piece of the biopsy (with negative results). One small testicular piece was processed for routine histology, a second one for meiotic studies with light microscopy, and a third piece was used for fine structural studies. Most of this piece was used for microspreading of synaptonemal complexes (SCs) from spermatocytes at pachytene, according to previously described methods (Solari, 1998). Another aliquot was fixed in 2% glutaraldehyde, post-fixed with 1% osmium tetroxide, embedded in Araldite and sectioned in thin (0.08 µm thick) and semi-thin (0.5 µm thick) slices for electron and light microscopy, respectively.
Electron microscopy of spread SCs and sections was made with a Siemens Elmiskop.
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Results |
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AZF microdeletions
Analysis of AZF genes revealed the presence of the three (A, B and C) regions. Thus, no microdeletion of the azoospermia locus in the Yq11 region was shown in this case.
Synaptonemal complex analysis
The analysis of the SCs at pachytene shows 20 autosomal SCs. In all the examined cells, a pentavalent and a univalent chromosome (frequently associated with the multivalent chromosome) are present (Figure 3). Thus, a complex rearrangement is present in spermatocytes. Electron micrographs of 20 different spermatocytes were analysed in prints. The pentavalent chromosome has four synapsed ends and two free ends. The special excrecences typical of the human X and Y axes in the normal XY body (Solari, 1980, 1988
, 1999
) allow the identification of the X axis and a segment of the Y axis. The X chromosome is always a terminal component of the pentavalent chromosome and is associated with another axis through a short SCwhich corresponds to the pseudoautosomal region (PAR). At the other end of the pentavalent, the terminal free end is rather short and is followed by the longest SC. This terminal axis is identified as one of the acrocentric chromosomes of the 1315 group because of its subterminal kinetochore location and the attachment of a nucleolar structure. The other two intermediate SCs are of similar length and should correspond to derivative chromosomes stemming from reciprocal translocations. In many spermatocytes, a small univalent is associated with this pentavalent at the free short end. The pentavalent and the relative lengths of the synaptic regions are shown in a diagram of the pachytene configuration (Figure 4).
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FISH with the X probe shows that the X chromosome is intact, as it paints only the submetacentric X chromosome and no signal is seen on any other chromosome.
The Y probe paints two chromosomes. One of them is the small marker chromosome that is painted on the heterochromatic arm, which is the same strongly DAPI+arm (Figure 5a). The second one is a mid-sized acrocentric chomosome that is painted on the proximal q region including the centromere (Figure 6a). The Y-alphoid DYZ3 repeat probe shows a signal in the centromeric region of this chromosome, and thus it is concluded that this is the chromosome that synapses with the X axis at pachytene through the PAR. Thus, this is a neo-Y chromosome, having the euchromatic part of the normal Y and an autosomal segment (Figure 6b).
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The PraderWilliAngelman probe shows one signal in the normal chromosome 15 and the other signal in the t(12;15) derivative chromosome (Figure 8).
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Discussion |
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In the present case, the use of the FISH technique was necessary for the correct diagnosis of this CCR, and the analysis of SCs was needed to guide the different FISH steps.
The patient shows the rare occurrence of a neo-Y chromosome, which is the result of a translocation of the euchromatic part of the Y chromosome on the long arm of chromosome 12. The signal of the terminus probe for 12p seen on the terminal region of the abnormal, long metacentric chromosome agrees with the assumption of a previous inversion of chromosome 12. Although in pachytene spreads an inversion loop was not consistently observed, it is known that such loops may rapidly disappear after early pachytene, leading to heterosynapsis. Thus, this complex chromosome rearrangement is apparently balanced and there are three chromosomes with four breakpoints (Figure 9): der(Y) (Ypter-Yq11.23::12q21.212qter); der(12)(12pter12p11.2::12q21.212p11.2::15q1315qter); and der(15)(15pter15q13::Yq11.23Yqter)
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The origin of pachytene apoptosis has been ascribed to a pachytene checkpoint (Roeder and Bailis, 2000) which detects failures in chromosome synapsis and recombination. This checkpoint may lead to pachytene arrest through p53-independent apoptosis (Odorisio et al., 1998
). In the present case, the long stretches of asynaptic regions in the pentavalent plus univalent at pachytene could initiate the apoptotic process. A further deleterious factor is the involvement of the sex chromosomes in this aberration and the close attachment of the autosomal regions of the pentavalent to the inactive X chromosome. The typical transcriptional silencing of the XY body in normal men (Solari, 1999
) may spread to close, autosomal chromatin (Jaafar et al., 1993
; Solari, 1999
), leading to the lack of transcripts necessary for completion of meiosis. The mechanisms of meiotic sex chromosome inactivation (MSCI) are presently unknown.
In this patient, the normal phenotype suggests that the breakpoints in Yq11.23, 12p11.2, 12q21.2 and 15q13 do not inactivate functional genes. The breakpoints may not include genes or gene regions with regulatory functions, whose disruption could produce phenotypic alterations. However, other disruptions at the molecular level cannot be disregarded.
The origin of CCRs is unknown. Exposure to ionizing radiation or the administration of immunosupressive drugs before or during pregnancy (Lucas et al., 1992), advanced paternal age (Benzacken et al., 1998
) and a possible instability of maternal chromosomes (Kousseff et al., 1987
) have been suggested as possible factors in the origin of CCRs.
In the present case, the paternal origin of the complex is evidenced by the presence of the Y-derived elements, and this paternal origin agrees with the previously recorded 11 cases of de novo CCRs. The literature shows that this is the second case of CCRs where the Y chromosome is involved. The other case (Joyce et al., 1999) was detected in a child with multiple congenital anomalies.
The scarce number of cases with male transmission were detected due to subfertility, oligospermia or azoospermia, as documented in the literature (Chandley et al., 1975; Joseph and Thomas, 1982
; Rodriguez et al., 1985
; Saadallah and Hulten, 1985
). Male carriers of CCRs may not be sterile, and the transmisssion of CCRs has been documented (Meer et al., 1981
; Bourrouillou et al., 1983
; Walker et al., 1985
; Gorski et al., 1986
; Schmidt and Passarge, 1988
). The genetic, reproductive risk of the CCR carriers depends on the involved chromosomes and the sites of breakpoints. The distribution of specific breakpoints is non-random, and frequent sites are located at 1q25, 4q13, 6q27, 7p14, 9q12, 11p11, 12q21, 13q31 and 18q21 (Gorski et al., 1988
). In general, the risk of spontaneous abortions is 48.3%, and 18.4% of all live births from CCR carriers resulted in phenotypically abnormal offspring (Gorski et al., 1988
; Lespinasse et al., 2003
).
In the present case, the theoretical origin of the CCR is interpreted as an initial inversion of chromosome 12 (at the breakpoints 12p11.2::12q21.2). The inverted 12 was translocated with chromosome 15, resulting in two derivative chromosomes, one of which is then involved in a further interchange with the Y chromosome (see Results). The scheme (Figure 10) shows the possible paths in the production of this CCR.
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Acknowledgements |
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References |
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Submitted on May 4, 2004; accepted on August 11, 2004.
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