DAZ Gene Research Articles

Investigation ofDAZandRBMY1Gene Expression in Human Testis by Quantitative Real-Time PCR

This study developed quantitative real-time PCR assays for the DAZ and RBMY1 genes to determine the copy number of RNA extracted from testicular biopsies from a cohort of normospermic controls (n=6) and azoospermic males (n=17) including two males with Y-chromosome microdeletions (AZFc and AZFb + c). All patients underwent testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI). Forty percent of the azoospermic cohort showed a significant reduction in the copies of at least one of the genes (DAZ P=0.003; RBMY1 P=0.009). The histopathology of these patients ranged from Sertoli cell only (SCO) to severe hypospermatogenesis with interstitial fibrosis. The patient with the AZFb + c deletion lacked expression of DAZ and RBMY1 and had a histopathology of SCO. The patient with the AZFc deletion had reduced expression of RBMY1 and no DAZ expression with a histopathology of spermatocyte arrest. The quantitative real-time PCR assays for DAZ and RBMY1 gave positive predictive values of 78% and 70%, respectively for the recovery of sperm from testicular biopsy.

O-243: Prevalence of AZF microdeletion (AZFb, AZFc and DAZ) in infertile males from the Korean populations

Most Y chromosome microdeletions occur on the long arm (q) and these microdeletions were mapped to three no overlapping regions, named as azoospermic factor (AZF). Deletions involving the AZFc region account for up to 90% of all Yq deletions with phenotypes varying from azoospermia to severe oligospermia and occasionally, mild oligozoospermia. The AZFc region, mainly DAZ gene cluster was the most frequently deleted region. The aim of this study was to evaluate the prevalence and type of Y microdeletions (AZFb, AZFc-DAZ) in Korean with male factor infertility.

DAZ gene copies: evidence of Y chromosome evolution

The DAZ gene, a contributing factor in infertility, lies on the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure. Y chromosome binary polymorphisms on the non-recombining Y (NRY) region, believed to be a single occurrence on an evolutionary scale, were typed in a sample of fertile and infertile men with known DAZ backgrounds. The Y single-nucleotide polymorphisms (Y-SNPs) with low mutation rates are currently well characterized and permit the construction of a unique phylogeny of haplogroups. DAZ haplotypes were defined using single-nucleotide variant (SNV)/sequence tagged-site (STS) markers to distinguish between the four copies of the gene. The variation of 10 Y chromosome short tandem repeat (STRs) was used to determine the coalescence age of DAZ haplotypes in a comparable time frame similar to that of SNP haplogroups. An association between DAZ haplotypes and Y chromosome haplogroups was found, and our data show that the DAZ gene is not under selective constraints and its evolution depends only on the mutation rate. The same variants were common to fertile and infertile men, although partial DAZ deletions occurred only in infertile men, suggesting that those should only be used as a tool for infertility diagnosis when analysed in combination with haplogroup determinations.

Association of spermatogenic failure with decreased CDC25A expression in infertile men

DAZ gene family is crucial for human spermatogenesis that requires the precise co-ordination of cell cycle events. CDC25A is recognized as the downstream substrate of DAZ gene family and is thought to function on the M-phase regulation of cell cycles. We investigated the expression profiles of CDC25A in the testes of infertile men and evaluated the relationship between CDC25A levels and testicular phenotype, clinical hormonal parameters and sperm retrieval results. The protein and mRNA transcript levels of CDC25A in the testes of 40 azoospermic men were determined by immunohistochemistry and quantitative real-time-PCR. CDC25A in human spermatozoa was investigated by western blotting and immunofluorescence staining. The CDC25A protein was expressed mainly in spermatocyte, spermatid and spermatozoa. CDC25A transcript levels were significantly decreased (P = 0.0009) in patients with spermatogenic failure, especially in men with meiotic arrest and Sertoli cell-only syndrome. Significantly higher CDC25A transcript levels were detected in patients with successful sperm retrieval than in patients with failed sperm retrieval (P = 0.005). Decreased CDC25A is associated with spermatogenic failure and failed sperm retrieval in infertile men. Further studies are necessary to explore the functional roles of CDC25A in human spermatozoa.

Quantitative evaluation of partial deletions of the DAZ gene cluster

Partial deletions of the DAZ gene cluster are thought to cause spermatogenesis impairment. The presence of homologous copies of this gene in the Y chromosome does not allow PCR to be used for the identification of this abnormality. Hence, sequence family variants (SFV), following amplification of sY581, sY587 and sY586 and subsequent enzymatic digestion with Sau3A, DraI and TaqI, respectively, and the dual fiber fluorescence in situ hybridization (FISH) have been used to this aim. However, SFV is not always able to identify single DAZ gene copy deletions. We report a quantitative real-time PCR application to evaluate partial deletions of the DAZ gene cluster. To accomplish this, we designed a probe on exon 6 of the DAZ gene which is repeated 3 times in DAZ1, once in DAZ2 and DAZ3 and twice in DAZ4. Five normozoospermic healthy men (C1-C5) having 4 DAZ gene copies by SFV were selected. Fiber-FISH confirmed this outcome in C1-C4, but not in C5 who had an incomplete DAZ gene cluster. The men underwent then quantitative real-time PCR and C1 was arbitrarily selected as calibrator for the calculation of the DAZ gene signals because of the lowest variation in the threshold cycles. Real-time PCR identified 7.2+/-0.05 signals in C2-C4 and 5.4+/-0.05 signals in C5. The overall coefficient of variation was 1.4+/-0.2%. The loss of two signals in this subject may relate to a deletion of both DAZ2 and DAZ3 or of DAZ4 gene. Since SFV showed clearly the presence of DAZ2, it may be hypothesized that C5 lacks DAZ4. In conclusion, these data suggested that quantitative real-time PCR seems to be an effective and reproducible technique that can be used to study the DAZ gene cluster. In addition, the probe chosen for this approach may give indication on the DAZ gene copy deleted.

Uniform deletion junctions of complete azoospermia factor region c deletion in infertile men in Taiwan

To determine the deletion junctions of infertile men in Taiwan with azoospermia factor region c (AZFc) deletions and to evaluate the genotype/phenotype correlation. Genomic DNAs from 460 infertile men were examined. Bacterial artificial chromosome clones were used to verify the accuracy of polymerase chain reaction. Deletion junctions of the AZFc region were determined by analysis of sequence-tagged sites and gene-specific markers. Complete AZFc deletions, including BPY2, CDY1 and DAZ genes, were identified in 24 men. The proximal breakpoints were clustered between sY1197 and sY1192, and the distal breakpoints were clustered between sY1054 and sY1125 in all but one of the 24 men. The testicular phenotypes of men with complete AZFc deletion varied from oligozoospermia, to hypospermatogenesis, to maturation arrest. We identified a group of infertile men with uniform deletion junctions of AZFc in the Taiwan population. Despite this homogeneous genetic defect in the AZFc region, no clear genotype/phenotype correlation could be demonstrated.

EVALUATION OF DAZ MICRODELETIONS IN 34 INFERTILE MEN

Microdeletions in Yq11 are a common molecular cause of spermatogenic failure in men and are recurrently detected in about 10-15% of idiopathic azoospermia and severe oligozoospermia. Screening for AZF microdeletions is often performed by multiplex PCR. AZFc deletions, involving the DAZ gene, form the majority of these deletions. The aim of this study was to evaluate in a group of 34 Tunisian infertile patients (16 oligozoospermic and 18 azoospermic men) the prevalence of DAZ microdeletions using a rapid molecular strategy: the PCR-DGGE method based on the high degree of homology between the DAZ gene and its autosomally equivalent DAZLA gene. DAZ microdeletions were detected in 8.8% of patients. The three deleted patients have a 46, XY karyotype. Two of them were azoospermic and the other had an extreme oligo-asthenoteratozoospermia with a predominant abnormality: small round head spermatozoa (Y46). Our findings suggest that PCR-DGGE method, for detection of DAZ gene deletion, could be particularly useful as a first step in the diagnosis workup of nonobstructive azoospermia and severe oligozoospermia for three reasons. First, it is a simple and fast system; second, DAZ microdeletions are the most common Y deletions; and third, partial DAZ microdeletions and mosaicism may be recognized by PCR-DGGE while only deletions removing the whole DAZ gene cluster can be detected by STS-PCR [211]. Nevertheless, this procedure has limitations because other deletions of AZFa and AZFb may go undetected. Therefore, molecular investigation by multiplex PCR must be conducted in a second step according to European guidelines for the molecular diagnosis of Y chromosome microdeletions, particularly before ICSI procedures.

Study on DAZ Gene Copy Deletion in Severe Oligozoospermia Sperm Donor for ICSI

Deletion of DAZ gene copies is related to spermatogenesis impairment. To investigate the distribution of DAZ gene copy deletions among Chinese men, we analyzed DAZ gene deletions by multiplex polymerase chain reaction (multi-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 128 infertile patients with severe oligozoospermia selected as semen donors for intracytoplasmic sperm injection (ICSI) and 287 normospermic men. Three patterns of DAZ gene deletions, namely DAZ1/DAZ2 deletion, DAZ3/DAZ4 deletion and complete deletion of all 4 DAZ copies, were found in the present study. Complete deletion of the entire DAZ family of genes was only present in 11.7% of severe oligozoospermic patients. The frequency of DAZ1/DAZ2 deletion was significantly higher in severe oligozoospermic patients than that in the controls(9.4% vs 2.8%, P = 0.004. The total frequency of complete DAZ deletion and DAZ1/DAZ2 deletion was 21.1%. No significant difference in the frequency of DAZ3/DAZ4 deletion was observed between the patient and control group (7.0% vs 3.8%, P > 0.05). These results suggest that complete DAZ deletion is a frequent genetic cause of severe oligozoospermia, and DAZ1/DAZ2 deletion is a high risk factor for the disease. Thus, it is necessary to screen the two deletion patterns of DAZ genes in severely oligozoospermic sperm donors before ICSI during assisted reproduction.

Genetic screening of the Dazl‐interacting protein genes

Abstract Micro‐deletions at specific loci of the Y chromosome have been observed frequently in male infertility patients, suggesting that genes in these regions are involved in male germ cell development. DAZ is a representative male infertility gene at the AZFc locus of the Y chromosome. Since DAZ contains an RNA binding motif along with so‐called a DAZ domain, it was proposed to participate in RNA metabolism during spermatogenesis. A mouse gene homologous to the human DAZ gene has been cloned and named Dazl (DAZ‐like). Dazl is autosomal and expressed in the testis and also at a low level in the ovary. Male mice homozygous for the Dazl null allele have small testes with a few spermatogonia and almost complete absence of germ cells beyond the spermatogonial stage, suggesting the requirement of Dazl for entry or progression through meiosis. However, its exact cellular functions have not been understood yet. In order to investigate cellular functions of Dazl, we decided to isolate candidate interacting protein genes of the mouse Dazl, using yeast two‐hybrid screening. A number of candidate Dazl‐interacting proteins have been isolated, such as Bprp, Acf, Hgs, Murr1, Nbak3 and Ranbp9, but dynein light chain 1 (Dlc1) was most predominant. A strong interaction of Dazl with Dlc1 suggests that Dazl might function as an mRNA adaptor to the dynein motor complex.

Dazl binds in vivo to specific transcripts and can regulate the pre-meiotic translation of Mvh in germ cells

Gametogenesis is a complex process subject to strict controls at both levels of transcription and translation. Members of a family of conserved RNA-binding proteins encoded by the DAZ genes are required for the translational regulation of gene expression essential for this process. Although loss of DAZ family genes is associated with infertility in several organisms including humans, the identity of the transcripts regulated in vivo is unknown. Using a combination of immunoprecipitation and microarray analysis, we have identified a number of mRNAs that are bound by the murine Dazl protein both in vivo and in vitro. Sequence analysis shows that these transcripts contain binding sites for Dazl, which have been conserved during evolution between human, rat and mouse. We have focussed on mouse vasa homologue (Mvh), a gene that is essential for male gametogenesis, and show that Dazl stimulates translation via the Mvh 3'-UTR. Finally, we show that germ cells of Dazl null mice contain reduced levels of Mvh protein, indicating that Dazl-mediated regulation of Mvh translation is crucial for mammalian spermatogenesis.

Copy number of DAZ genes in infertile men

The aim of the study was to define the relevance of deletions and duplications within the DAZ gene cluster to male factor infertility in a population of 90 infertile men and a control of 50 fertile men using real-time polymerase chain reaction (PCR). We conclude that partial deletions of the DAZ genes are associated with oligozoospermia but not with azoospermia; however, an increased number of DAZ genes does not seem to be a statistically significant risk factor for spermatogenic failure.

13p and Yq Homology Have Anything To Do With Male Infertility Status: A Novel Familial Inheritance of 13p Deletion

13p and Yq Homology Have Anything To Do With Male Infertility Status: A Novel Familial Inheritance of 13p Deletion

The Y chromosome gr/gr subdeletion is associated with male infertility

Men with Y chromosome (Yq) AZFc deletions lack all copies of the DAZ gene and have severe spermatogenic failure. A recently described gr/gr subdeletion of AZFc removes two of four copies of DAZ. To better understand the relative frequencies of AZFc and gr/gr deletions and their associated phenotypes, we analysed two large groups of infertile men. A total of 788 men from the Monash Male Infertility (MMI) database with a range of fertility disorders showed similar overall prevalences of AZFc (2.5%) and gr/gr deletions (3.4%). There was no association of gr/gr deletions with sperm density. In 234 control men of known or presumed fertility, only one gr/gr deletion was found. In a further 599 consecutive men presenting for assisted reproductive technologies, we detected 13 (2.2%) AZFc deletions and 28 (4.7%) gr/gr deletions. All AZFc deletions were seen with sperm densities <5 million/ml but again the gr/gr deletion occurred with similar frequency across all sperm density categories. These data show that gr/gr deletions are significantly associated with infertility in the Australian population (P = 0.0015) but not exclusively with reduced sperm density suggesting a complex interaction with other factors important for male fertility. Vertical transmission of gr/gr deletions from father to son by ICSI was demonstrated in four cases. Analysis of 130 ICSI-conceived sons revealed no de novo gr/gr deletions indicating that ICSI is not a risk factor. The data suggest that testing for gr/gr deletions should be considered in the routine genetic assessment of men with idiopathic infertility.

No partial DAZ deletions but frequent gene conversion events on the Y chromosome of fertile men

Recently, partial DAZ deletions on the Y chromosome were identified in infertile men. To determine the clinical importance of partial DAZ deletion, we studied the number of DAZ copies in a well-defined population of 47 fertile men. The number of DAZ gene copies was determined by PCR assays, qualitative and quantitative DNA blot experiments. Using semi-quantitative Southern blot, no partial DAZ deletion was detected in fertile men. In many cases, the results were discordant with the PCR assays and qualitative DYS1-blot experiments suggesting that the molecular events detected by the later methods could reflect gene conversion events. Many fertile men present four copies of the DAZ genes but an atypical organization of this DAZ locus. No difference in sperm concentration and motility in the fertile men were observed according to the different DAZ-haplotypes. The different DAZ-haplotypes are compatible with normal spermatogenesis.

Unique t(Y;1)(q12;q12) reciprocal translocation with loss of the heterochromatic region of chromosome 1 in a male with azoospermia due to meiotic arrest: a case report

A de novo reciprocal translocation 46,X,t(Y;1)(q12;q12) was found in an azoospermic male with meiotic arrest. Cytogenetics and fluorescent in situ hybridization (FISH) were used to define the karyotype, translocation breakpoints and homologue pairing. SRY (Yp), Yq11.2-AZF regions, DAZ gene copies and the distal Yq12 heterochromatin were studied by PCR and restriction analysis using sequence-tagged sites and single nucleotide variants. High resolution GTL, CBL and DA-DAPI staining revealed a (Y;1) translocation in all metaphases and a normal karyotype in the patient's father. FISH showed the presence of the distal Yq12 heterochromatic region in der(1) and loss of the heterochromatic region of chromosome 1. PCR demonstrated the intactness of the Y chromosome, including the SRY locus, AZF regions, DAZ genes and distal heterochromatin. A significant decrease (P = 0.005) of Xp/Yp pairing (18.6%), as compared with controls (65.7%), was found in arrested primary spermatocytes, and cell culture and mRNA expression studies confirmed an irreversible arrest at meiosis I, with induction of apoptosis and removal of germ cells by Sertoli cells. We characterized a de novo t(Y;1)(q12;q12) balanced reciprocal translocation with loss of the heterochromatic region of chromosome 1, that caused unpairing of sex chromosomes followed by meiosis I arrest, apoptotic degeneration of germ cells and azoospermia.

Association of partial AZFc region deletions with spermatogenic impairment and male infertility

Background: Complete deletions of the AZFc region in distal Yq are the most frequent molecular genetic cause of severe male infertility. They are caused by intrachromosomal homologous recombination between amplicons—large,...

Role of the DAZ genes in male fertility

Males contribute to about 50% of infertility in humans and Y chromosome deletions are the major known genetic contribution to this. Amongst the genes encompassed by these deletions are the DAZ genes. The DAZ family of genes (consisting of homologues of BOULE, DAZL and DAZ) encode highly conserved RNA-binding proteins that are essential for gametogenesis in metazoans. They join the ranks of proteins that act to control this complex developmental process by regulating the translation of specific mRNAs. Advances in knowledge of how this gene family acts to regulate key meiotic events in model organisms will lead to a fuller understanding of their function in human fertility.

High frequency of gr/gr chromosome Y deletions in consecutive oligospermic ICSI candidates

The Y chromosome gr/gr microdeletion eliminates two copies of the DAZ gene and several additional transcriptional units and has been associated as a risk factor for infertility. Our objective was to study the presence of the gr/gr deletion in ICSI candidates in our population and to determine whether the laboratory, clinical and ICSI outcome were different in the gr/gr deleted patients. Two hundred and eighty-three ICSI candidates were studied. Semen analysis, serum FSH, LH, testosterone, inhibin B, karyotype and detection of sequence tagged sites in the Y chromosome were performed. gr/gr deletions were detected in 11 (5.07%) of 217 oligospermic and in one (1.52%) of 66 azoospermic consecutive ICSI candidates, but in none of 232 controls (P=0.002). The fertility rate was not different in the four patients of the gr/gr deleted group treated by ICSI (64.38%; 47/73) as compared to average results at our center (65.49%; 2393/3654). gr/gr deletions are a risk factor for spermatogenic failure at our population, but the prognosis of the four patients of the gr/gr deleted group treated by ICSI is not different from that of other ICSI patients.

No association of the A260G and A386G DAZL single nucleotide polymorphisms with male infertility in a Caucasian population

The human DAZ gene family includes two autosomal genes, BOULE and DAZL, and a Y-chromosomal DAZ gene cluster. All are RNA-binding proteins and assumed to be master regulators of germline gene expression. We have investigated the impact of two DAZL polymorphisms, located at nucleotide positions 260 (SNP 260) and 386 (SNP 386), on the fertility of Caucasian men. These single nucleotide polymorphisms (SNPs) have been described previously to be associated with spermatogenic failure. Blood samples were collected and genomic DNA was extracted from 165 normozoospermic men and 202 oligo- or azoospermic patients, of whom 28 displayed an AZFc deletion. The frequencies of A or G allelic variants in SNP 260 and 386 were analysed via TaqMan allelic discrimination assays. In both cases, the A to G transition leads to a threonine to alanine change. A total of 24.2% of the controls showed a heterozygous nucleotide variant (AG) for the SNP 260 and the remaining 75.8% were homozygous for A. In the AZFc-deleted group, this distribution was significantly different, with 39.3% for AG, 57.1% for AA and 3.6% for GG. However, the increased heterozygosity was not correlated with sperm counts and morphology. The patients without deletions displayed a similar allelic pattern to the controls (24.1% AG/75.9% AA). For SNP 386, only the AA nucleotide variant was found in all subjects studied and in no case was the previously described heterozygous AG variant found. In a selected Caucasian population, the DAZL SNP 386 is completely absent and SNP 260 is not associated with spermatogenic failure and therefore does not represent a molecular marker for genetic diagnosis of male infertility.

AZF and DAZ gene copy-specific deletion analysis in maturation arrest and Sertoli cell-only syndrome.

Deletions of the AZFc region in Yq11.2, which include the DAZ gene family, are responsible for most cases of male infertility and were associated with severe oligozoospermia and also with a variable testicular pathology. To uncover the functional contribution of DAZ to human spermatogenesis, a DAZ gene copy-specific deletion analysis was previously established and showed that DAZ1/DAZ2 deletions associate with oligozoospermia. In this study we applied the same screening method to 50 control fertile males and 91 non-obstructive azoospermic males, 39 with Sertoli cell-only syndrome (SCOS) and 52 with meiotic arrest (MA). Samples were also screened with 24 sequence-tagged sites to the different AZF regions, including 114 control fertile males. After biopsy (testicular sperm extraction, TESE), residual spermiogenesis was found in 57.7% MA and 30.8% SCOS cases (incomplete syndromes). DAZ1/DAZ2 deletions were associated with the testicular phenotype of residual spermiogenesis as they were only found in two patients (8%) with incomplete MA. Differences between incomplete (23.3%) and complete (4.5%) MA cases regarding AZFc and DAZ1/DAZ2 deletion frequencies, and between incomplete (58.3%) and complete (11.1%) SCOS cases for AZFc deletions, suggest that incomplete syndromes might represent an aggravation of the oligozoospermic phenotype. As successful TESE was achieved in 87.5% of MA cases with AZFc and DAZ1/DAZ2 deletions and in 58.3% of SCOS cases with AZFc deletions, the present results also suggest that these molecular markers might be used for the establishment of a prognosis before TESE.