Molecular Human Reproduction, Vol. 9, No. 4, 183-188,
April 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
The use of spermHALO-FISH to determine DAZ gene copy number
Submitted on December 12, 2002; accepted on January 1, 2003
1 Center for Reproductive Medicine and Department of Obstetrics and Gynaecology and 2 Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands
3 To whom correspondence should be addressed at: Center for Reproductive Medicine (A1-230), Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. e-mail: S.Repping{at}amc.uva.nl
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ABSTRACT |
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The AZFc region of the human Y chromosome is frequently deleted in men with spermatogenic failure and contains many multicopy genes. The best-characterized gene family within this region is the Deleted in AZoospermia (DAZ) gene family, which is present in four nearly identical copies. Recent reports claim deletions of some but not all DAZ genes. The assays used in these studies, however, are unable to provide conclusive evidence on the number of DAZ genes. In this study we show that with the use of highly decondensed sperm nuclei with large DNA domains (spermHALO) it is possible to determine the number of DAZ genes accurately. Using this fluorescent in-situ hybridization (FISH) technique, which has both high resolution and high range, we show that in 10 normospermic men, in which PCR digest assays indicated a deletion of one or more DAZ genes, all four DAZ genes were present. Also we confirmed previous findings of a deletion of two DAZ genes in two men and identified a man with six DAZ genes. Our results indicate that spermHALO-FISH allows an accurate determination of DAZ gene copy number, while PCR digest assays do not. Therefore, confirmation of positive results from PCR digest assays with spermHALO-FISH is essential. Furthermore, the spermHALO-FISH technique should prove useful as a genetic mapping technique in other regions of the Y chromosome and similar repetitive regions throughout the genome.
Key words: AZFc/DAZ/FISH/gene copy number/male infertility
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Introduction |
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Deletion of Y-chromosomal DNA is the most common molecular cause of spermatogenic failure in men. Four common classes of such deletions have been described: AZFa, P5/proximal P1 (AZFb), P5/distal P1, and AZFc deletions (Vogt et al., 1996; Repping et al., 2002). Deletions of the AZFc region are found in
10% of men with azoospermia (Reijo et al., 1996; Kremer et al., 1997; Kuroda-Kawaguchi et al., 2001). The AZFc region contains seven gene families with a total of 18 gene copies that are all expressed exclusively or predominantly in the testis (Kuroda-Kawaguchi et al., 2001). One of these genes is the Deleted in Azoospermia (DAZ) gene. Although the precise function of DAZ is unclear, studies in several species indicate that disruption of this gene leads to spermatogenic failure (Eberhart et al., 1996; Ruggiu et al., 1997; Houston and King, 2000). DAZ was originally thought to be single copy, but the presence of slight differences in sequenced cosmids from a single man indicated that there were at least two copies (Reijo et al., 1995; Saxena et al., 1996). Subsequently, several studies claimed that there were anywhere between three and seven copies of the gene (Glaser et al., 1997; 1998; Yen et al., 1997; Yen, 1998). With the use of conventional fluorescence in-situ hybridization (FISH) techniques and the complete nucleotide sequence of the AZFc region, we now know that in fact there are four DAZ genes on the human Y chromosome, arranged in two clusters with two genes in head-to-head orientation (Saxena et al., 2000; Kuroda-Kawaguchi et al., 2001).
Recently, two studies claim to have found deletions of some but not all copies of the DAZ genes in subfertile men, suggesting such deletions as causes of spermatogenic failure (de Vries et al., 2002a; Fernandes et al., 2002). Consequently, the search for variation in DAZ gene copy number and the role of such variation in spermatogenic failure is ongoing. However, determining the precise number of DAZ genes and searching for possible deletions of some but not all gene copies is difficult. The reason is that members of gene families on the human Y chromosome, such as DAZ, show relatively few differences in nucleotide sequence thereby precluding the use of PCR assays to allow distinction of the different copies.
To elucidate the DAZ gene copy number and to search for possible deletions of some copies of DAZ, two methods are available: PCR digest assays, and FISH techniques. The PCR digest assays make use of the subtle nucleotide variations in different copies of DAZ, so-called sequence family variants (SFV) or single nucleotide variants (SNV). However, a drawback of these PCR digest assays is that the variants studied appear to be polymorphic, making interpretation of the results difficult (de Vries et al., 2002a).
FISH, on the other hand, is a powerful tool in highly repetitive regions such as AZFc. By using DAZ-specific cosmids as probes and leukocyte interphase nuclei as targets (IP-FISH), it is possible to determine the number of DAZ gene clusters. Fibre-FISH, which makes use of extended chromatin fibres from leukocytes, allows the determination of the number of genes within a cluster. Although useful, a drawback of these two techniques is that it is impossible to visualize directly both clusters with all four genes in a single microscopic image. Consequently, a deletion of a single DAZ gene copy cannot be excluded with these techniques.
Here we aim to provide conclusive evidence on DAZ gene copy number by using spermHALO—highly decondensed sperm nuclei with large DNA loop domains—as a template for FISH. Furthermore, we compare the results from spermHALO-FISH with results from conventional FISH techniques and PCR digest assays.
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Materials and methods |
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Patient samples
In total we investigated 22 fertile semen donors who participated in our programme for artificial insemination with donor semen and six subfertile patients who were previously identified as having a reduced DAZ gene copy number by SFV and conventional FISH techniques (de Vries et al., 2002a). From each man, blood was drawn for isolation of DNA and isolation of leukocytes. A semen sample was obtained and used for spermHALO preparations as described below.
This study was approved by the Institutional Review Board of the Academic Medical Center and all donors and patients gave informed consent.
PCR digest assays
Analysis of SFV was performed using markers sY581, sY586 and sY587 as described previously (see Table I) (Saxena et al., 2000; de Vries et al., 2002a). Analysis of SNV and of DAZ-RRM3 and Y-DAZ3 was performed according to Fernandes et al. (2002) (see Table I).
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SpermHALO preparations
To overcome the limitations of IP-FISH and fibre-FISH, we applied a different method using highly decondensed sperm (spermHALO) as a template for FISH. By exposing sperm to dithiothreitol (DTT), a gradual swelling of the nucleus is achieved with chromatin fibres extending from the sperm head while remaining attached to the nuclear annulus (see Figure 1) (Ward et al., 1989). These DNA domain loops consist of linear DNA and therefore provide optimal resolution for FISH analysis. Furthermore, one can readily observe the entire cell in a single microscopic image without confounding effects of neighbouring cells (Figure 2D).
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SpermHALO were prepared from semen samples that were obtained by masturbation after
2 days of sexual abstinence and subsequently cryopreserved (for the subfertile men we used fresh ejaculate instead of frozen samples). After thawing, sperm were separated from seminal plasma by washing twice with 10 mmol/l Tris–HCl (10 min, 300 g). Subsequently, the pellet was resuspended in 5 ml 0.75% sodium dodecyl sulphate (SDS), 10 mmol/l Tris–HCl, mixed, incubated for 5 min at room temperature, and mixed again. The SDS solution was removed after centrifugation (10 min, 300 g) and the pellet was washed with 10 mmol/l Tris–HCl (10 min, 300 g). The pellet was then diluted in 10 mmol/l Tris–HCl to a concentration of 25x106 sperm per ml. The sample was diluted 1:9 in extraction buffer (2 mol/l NaCl, 10 mmol/l DTT, 10 mmol/l Tris, pH 7.4) and incubated for 0.5 to 4 min at 37°C (the duration of incubation varied for different patients as the response to the extraction buffer was variable; also, the concentration of DTT was adjusted for some semen samples because of under- or overswelling of the sperm nuclei in these samples). After extraction, small droplets of the samples were dropped on ice-cold slides and incubated at 0°C for 25 min. Subsequently, the extraction buffer was removed by gently dropping 10 mmol/l Tris–HCl onto the slides. Finally, slides were air-dried and dehydrated using a 70, 96 and 100% ethanol series. Slides were used immediately for FISH analysis.
FISH
One or two-colour FISH was performed as described previously (Saxena et al., 2000; de Vries et al., 2002a). Slides with either interphase nuclei (IP-FISH), extended chromatin fibres (fibre-FISH) or highly decondensed sperm nuclei with large DNA domains (spermHALO-FISH) were used as hybridization targets. Two sequenced DAZ cosmids were used as probes: cosmid 18E8 (encompassing both 5' ends and the intervening sequence of two neighbouring DAZ genes) and cosmid 46A6 (encompassing the 3' end and 35 kb of flanking sequence). To determine the number of DAZ clusters with the use of IP-FISH, the number of signals for probe 18E8 was counted in 200 nuclei. For fibre-FISH and spermHALO-FISH, the number of DAZ genes was determined by examining the order of the signals (which appear as beads-on-a-string) after two-colour hybridization with both DAZ probes.
Accession numbers
GenBank accession numbers (http://www.ncbi.nlm.nih.gov/Genbank) are as follows. sY581: G63906; sY586 = SNV III: G63907; sY587 = SNV V: G63908; SNV I: G73167; SNV II: G73163; SNV IV: G73168; SNV VI: G73169; Y-DAZ3: G73170; DAZ-RRM3: G73171.
Online Mendelian Inheritance in Man (OMIM) (http://www.ncbi.nlm.nih.gov/Omim/): for AZFc: MIM 40024.
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Results |
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PCR digest assays
Analysis of PCR digest assays in the 22 fertile semen donors gave a diversity of results. Interpretation of these results suggested a deletion of at least one DAZ gene in 10/22 men examined. One man appeared to have a deletion of DAZ3 only, eight men showed PCR digest results suggesting a deletion of DAZ2 and DAZ4 and one man appeared to have retained only DAZ1 (Table II). All six patients who were previously identified as having a reduced DAZ gene copy number by SFV and conventional FISH techniques, showed aberrations in SNV as well. More precisely, the results indicated a deletion of two genes in one man, a deletion of three genes in four men and in one man PCR digest results could be interpreted as the absence of all four DAZ genes (Table III).
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Conventional FISH techniques
In contrast to the large number of aberrations found with PCR digest assays, IP-FISH using probe 18E8 showed two signals, representing two DAZ gene clusters, in all but donor ID20 (see Figure 2A). In this donor, who appeared to be deleted for DAZ2 and DAZ4 by SNV analysis, IP-FISH showed three signals in the majority of nuclei examined indicating the presence of three DAZ gene clusters (Figure 2B, C). The presence of three DAZ gene clusters in this donor had been noted before in a study that determined the number of DAZ gene clusters in sperm nuclei (de Vries et al., 2002b). IP-FISH in the six patients was as reported previously, i.e. all patients had only one DAZ cluster (de Vries et al., 2002a).
Fibre-FISH analysis using extended chromatin fibres from leukocytes showed the presence of two DAZ genes in head-to-head orientation in all donors, including the donor with three clusters (Figure 2d). Again, fibre-FISH in the six patients was as reported previously, i.e. all patients had two DAZ genes in head-to-head orientation (de Vries et al., 2002a).
SpermHALO-FISH
We were able to determine the DAZ gene copy number in all donors and in both patients for whom sperm were available (from four patients no sperm were available; see Table III). In 21 donors we clearly observed four DAZ genes arranged in two clusters with two genes in head-to-head orientation (Table II, Figure 3A). Similarly, we observed only a single DAZ cluster with two genes in both patients previously identified as having a reduced DAZ gene copy number (Table II, Figure 3B). Finally, spermHALO from donor ID20, in which IP-FISH showed three DAZ clusters, showed the presence of six DAZ genes organized in three identical clusters with two genes in head-to-head orientation (Figure 3c).
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Discussion |
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In this study we showed that spermHALO-FISH allows the precise determination of DAZ gene copy number. In contrast, PCR digest assays gave false positive results in 10 men. In nine of these men spermHALO-FISH clearly showed the presence of four DAZ copies, and in one man a total of six DAZ genes were found. We also showed that all but one sperm donor carried four DAZ copies and confirmed previous findings in two patients who were deleted for two DAZ genes.
All four common classes of Y chromosome deletions that have been described to date result in the deletion of all copies of at least two gene families (Blanco et al., 2000; Kamp et al., 2000; Sun et al., 2000; Kuroda-Kawaguchi et al., 2001; Repping et al., 2002). However, not much is known about deletions involving only some of the genes of a Y-chromosome gene family. Recently, two reports have described the deletion of some of the four DAZ genes in a group of subfertile men (de Vries et al., 2002a; Fernandes et al., 2002). Our results from spermHALO-FISH here further confirm this finding. The main drawback in searching for such deletions is the lack of an appropriate screening technique. Investigators have tried to use subtle nucleotide differences between different gene copies of the same gene family to search for such deletions. Although relatively fast, these techniques often result in false-positive findings as shown in this study. While all men with a reduced DAZ gene copy number were detected with PCR digest assays (100% sensitivity), the specificity of PCR digest assays was only 38% (only 6/16 men with PCR digest assay aberrations had indeed a deletion of two DAZ genes). Also, in four of the six men with only two DAZ genes, PCR digest assays indicated that there was only one DAZ gene present. One man (AMC0135) had aberrations in eight of the nine PCR digest assays that when interpreted together could suggest the absence of all four DAZ genes; again, spermHALO-FISH showed the presence of two DAZ genes.
FISH techniques are apparently far more reliable than PCR digest assays, but require expertise and are laborious. IP-FISH allows the determination of the number of DAZ gene clusters, while fibre-FISH can subsequently determine the number of genes within such a cluster. These techniques, however, cannot rule out the deletion of a single DAZ gene. On the other hand, the technique of spermHALO-FISH as described here, proved to be highly useful in determining the precise DAZ gene copy number. This technique has previously been used to examine the structure and cellular function of the sperm nuclear matrix, but not as a genetic mapping technique (De Lara et al., 1993; Choudhary et al., 1995; Nadel et al., 1995; Yaron et al., 1998; Schmid et al., 2001). The highly decondensed DNA loop domains provide both high resolution and high range (several Mb), thereby forming an ideal template for FISH. Furthermore, due to the attachment of the DNA loop domains to the nuclear annulus, one can easily identify the total amount of DNA originating from a single cell, thereby eliminating confounding effects from neighbouring cells.
The fact that PCR digest assays are not highly reliable in determining DAZ gene copy number indicates that the nucleotide differences on which these techniques rely are polymorphic. The absence of one of the two variants apparently does not indicate the absence of the gene that contains the variant but more likely means that both genes contain the same variant. Recent reports indicate that Y–Y gene conversion probably underlies the polymorphic nature of these variants (S.Rozen et al., unpublished data; H.Skaletsky et al., unpublished data). Gene conversion occurs when homologous recombination between repetitive structures, which are abundant on the human Y chromosome, gives rise to unidirectional copying (Figure 4). In the absence of meiotic recombination with a sister chromosome, this process seems to be highly frequent on the Y chromosome (S.Rozen et al., unpublished data). Our results here show that the degree of variation differs between different nucleotide variants, ranging from zero (markers SNV-II, sY581, sY587 and DAZ-RRM3) to nine (marker SNV-VI) out of 22 samples examined (Table II). This variation could indicate that some sites are more susceptible to gene conversion events than others or that some of these variants have occurred quite recently in evolution.
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As a result of these gene conversion events, it is easy to misinterpret the absence of a subtle nucleotide variant as a deletion event. Therefore, when finding aberrations with assays that rely on these polymorphic differences, spermHALO-FISH (or, when not possible, conventional IP-FISH and fibre-FISH) should be performed to confirm or contradict these findings.
The technique of spermHALO-FISH is highly reliable in determining DAZ gene copy number and provides a useful tool in searching for deletions of some but not all of the DAZ genes in subfertile men. Besides its use for determining DAZ gene copy number, it should also be useful in determining gene copy number of other multicopy genes on the Y chromosome. Furthermore, its high resolution and high range make it a potentially useful method for clarifying the genomic structure in other repetitive regions throughout the genome.
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Acknowledgements |
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We thank Laura Brown and David C.Page for the use of cosmids 18E8 and 46A6.
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