Human DAZL1 encodes a candidate fertility factor in women that localizes to the prenatal and postnatal germ cells

David M.Dorfman1, David R.Genest1 and Renee A.Reijo Pera2,3

1 Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 0211 and 2 Department of Obstetrics, Gynecology and Reproductive Sciences, and Department of Physiology, University of California, San Francisco, CA 94143–0720, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 
The human DAZ (Deleted in AZoospermia) gene family contains a cluster of DAZ genes on the Y chromosome and a single autosomal homologue, DAZL1 (DAZ-Like) that maps to chromosome 3p24. Although the role of the Y chromosome gene family in determining fertility and the expression of the gene family has been well explored in men, little is known of the role of the DAZL1 gene in determining the fertility of women. In mice, loss of function of the homologue of DAZL1 results in the loss of male and female germ cells. In mice, Dazl1 protein is localized to prenatal and postnatal follicles. Here we demonstrate using two antisera that recognize human DAZL1 that the protein is expressed embryonically in germ cells of girls and in mature oocytes. This pattern of expression suggests that the DAZL1 gene is a candidate fertility factor in women and that it would be appropriate to search for mutations in the DAZL1 gene in peripheral blood DNA from women with primary amenorrhoea or premature ovarian failure.

Key words: /female fertility/premature ovarian failure/primary amenorrhoea


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 
The human DAZ (Deleted in AZoospermia) genes were identified in a search for Y chromosome genes that cause infertility in men. Y chromosome deletions encompassing the DAZ genes are the most common molecularly defined cause of infertility in men (Reijo et al., 1995Go). Y chromosome DAZ genes are found only in primates, whereas autosomal homologues of the DAZ genes are found in diverse organisms including flies, worms, frogs, and all mammals including humans (Reijo et al., 1995Go, 1996aGo; Cooke et al., 1996Go; Maiwald et al., 1996Go; Saxena et al., 1996Go; Yen et al., 1996Go; Carani et al., 1997Go; Chai et al., 1997Go; Karashima et al., 1997Go; Seboun et al., 1997Go; Houston et al., 1998Go). Loss of the region of the Y chromosome encompassing the DAZ gene cluster in humans is characterized by the presence of few or no germ cells frequently with meiotic arrest (Reijo et al., 1995Go, 1996bGo; Pryor et al., 1997Go). Loss of function of the gene homologous to DAZ in flies leads to meiotic arrest in males (Eberhart et al., 1996Go), whereas loss of function of the gene homologous to DAZ in Caenorhabditis elegans leads to meiotic arrest in females (Karashima et al., 1997Go). Interestingly, loss of function of the mouse gene homologous to DAZ, Dazl1 (Deleted in azoospermia-like), leads to both male and female infertility characterized by prenatal loss of germ cell populations (Ruggiu et al., 1997Go). In the frog, Xenopus laevis, nothing is known of the genetic requirement for a DAZ/DAZL1 homologue, but it is clear that a member of this gene family is expressed in the germ plasm that ultimately gives rise to the entire germ cell lineage. Frog DAZ homologue is also expressed in adult germ cells (Houston et al., 1998Go).

In recent reports, it was suggested that in male mice Dazl1 protein is restricted to the cytoplasm of spermatocytes, and that human DAZ protein is restricted to postmeiotic male germ cells (Ruggiu et al., 1997Go; Habermann et al., 1998Go). In contrast, we found that the human DAZ and DAZL1 proteins were present in both the nucleus and cytoplasm of male gonocytes and were abundant in the nuclei of spermatogonia. At meiosis, both proteins relocated to the germ cell cytoplasm (R.Reijo et al., unpublished results). These results, together with findings in diverse species, suggest that proteins encoded by the DAZ family may function in multiple cellular compartments and at multiple points in male germ cell development. They may act during meiosis and much earlier, during the establishment of spermatogonial stem cell populations (R.Reijo et al., unpublished results).

All of the studies on the DAZ family of genes in humans have focused on fertility in men. Nothing is yet known about the role of the DAZL1 gene in determining fertility in women even though homologues are required for female fertility in other organisms (Karashima et al., 1997Go; Ruggiu et al., 1997Go). In this report, we investigated the localization of the human DAZL1 protein in female tissues. It is critical that we examine expression of the human gene in women if we are to predict what phenotypes might be associated with mutations in DAZL1 in women.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 
Reverse transcription–polymerase chain reaction (RT–PCR)
RT–PCR analysis of DAZL expression was examined in cDNA from several tissues (mRNA were obtained from Clontech, Inc., Palo Alto, CA, USA). Primer sequences were as previously reported (Saxena et al., 1996Go); left primer: GGAGCTATGTTGTACCTCC; right primer: GTGGGCCATTTCCAGAGGG. These primers are specific for the DAZL1 cDNA. They do not recognize the DAZ cDNA since the right sequence is deleted from the DAZ cDNA. RT–PCR was performed on cDNA in a reaction that contained 50 ng of cDNA, 2 µl of 10X Promega Taq Polymerase buffer (Promega, Inc., Madison, WI, USA), 2 µl of 1 mmol/l dNTP, 0.5 units of Taq polymerase, 1 µl of each primer at 20 µmol/l concentration in a 20 µl reaction. Thermal cycling was as follows: 94°C for 5 min followed by 35 cycles of 94°C, 1 min; 60°C, 1 min; 72°C, 1 min. Products were run on a 4% NuSieve gel. The expected product size for the DAZL1 cDNA is 128 bp. To control for non-specific priming, reactions were run with and without inclusion of the reverse transcriptase enzyme. Since authentic cDNA is produced only in the presence of reverse transcriptase, bands produced without this enzyme are considered spurious.

Preparation of anti-DAZ/DAZL antibodies and Western blotting
Two antibodies were prepared in duplicate in rabbits (Research Genetics, Inc., Huntsville, AL, USA) against peptide sequences that are specific to DAZ/DAZL1 protein family members. Prior to injection of the peptides into rabbits, they were coupled to an MAP peptide (a branched lysine structure). After 10 weeks, specificity of antibodies was initially tested by Western blotting. Then, serum antibodies were affinity-purified on Protein A columns according to manufacturer's instructions (Hi-Trap; Pharmacia, Inc., Uppsala, Sweden). Antibodies were preabsorbed on mouse liver acetone powder that had been prewashed with phosphate-buffered saline (PBS) (three times) to remove non-specific binding activity before further use. For Western blots, proteins from tissue samples were lysed in 10 vol sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis buffer containing 100 mmol/l Tris–Cl (pH 6.8), 200 mmol/l dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol by heating the samples to 95°C for 5 min. The samples were then placed on ice, sonicated vigorously for 20 s to shear DNA, and 20 µg of protein extract were run on a 12% acrylamide gel. Proteins were transferred to nitrocellulose filters using a BioRad transfer system for 1 h at 100 V. The nitrocellulose filters were incubated in a 5% solution of milk powder in Tris-buffered saline (TBS) containing 0.1 mol/l Tris–HCl, pH 7.5, 0.9% NaCl, and 0.2% nonidet 40 (NP40) for 1 h to block non-specific binding of antibodies to the filters. The filters containing transferred proteins were then incubated in a 1:1000 dilution of primary antisera 149, 150, or preimmune for 1 h at room temperature followed by three washes, 5 min each, with TBS, milk, and NP40 buffer as above. After incubation with a 1:10 000 dilution of secondary antibody, anti-rabbit IgG–peroxidase conjugate (Sigma Chemical Co., St Louis, MO, USA) in the above buffer, the filters were washed twice in buffer as described above, once in TBS, milk plus 0.1% Tween-20, and twice in TBS for 5 min each rinse. The peroxidase reactions were visualized using the electrochemiluminescence (ECL) system according to manufacturer's instructions (Amersham, Inc., Arlington Heights, IL, USA).

Immunohistochemical localization of DAZL protein in human ovary
The localization of the DAZL protein was determined using preimmune antisera (a, b), antiserum 149 which recognizes the conserved RNA binding domain of all DAZ/DAZL family members (c,d) and antiserum 150 which recognizes a domain specific to the carboxyterminus of DAZL (e, f). Tissue sections are from fetal ovary, 19 weeks gestation (a, c, e) and adult ovary from a 37 year old female (b, d) and a 44 year old female (f). Formalin-fixed, paraffin-embedded human tissues were sectioned at 5 µm for immunoperoxidase staining. After baking at 60°C for 1 h, sections were deparaffinized and rehydrated (100% xylene x4 at 3 min each, 100% ethanol x2 at 3 min each, 95% ethanol x2 at 3 min each, water rinse for 5 min). Slides were then blocked with 3% hydrogen peroxide in absolute methanol for 5 min at room temperature, washed with water for 5 min, and microwave-treated (800 W, General Electric) at 199°F for 30 min in pre-heated 10 mmol/l citrate buffer, pH 6.0 (gently agitated every 5 min). Slides were cooled for 15 min, transferred to PBS + 0.1% Tween-20, and treated with 1.5% goat serum for 15 min at room temperature. Slides were incubated with 149 antibody (1:4000) or 150 antibody (1:1000) in 2% goat serum for 1 h at room temperature, washed in PBS + 0.1% Tween20 3x5 min each, incubated with biotinylated horse anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature, washed with PBS + 0.1% Tween-20, then incubated with avidin–biotinylated peroxidase complex (Vector) for 40 min at room temperature, followed by reaction with DAB/hydrogen peroxide. Sections were subsequently stained with 2% Gill's haematoxylin. Column-fractionated preimmune sera corresponding to fractions containing the specific antibodies were used as negative controls.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 References
 
RT–PCR of DAZL1 cDNA from human tissues
We first explored the distribution of DAZL1 messenger RNA by RT–PCR on mRNA from several tissues including heart, liver, ovary, testis and brain using primers that amplify the DAZL1 gene and cDNA specifically. As shown in Figure 1aGo, the DAZL1 gene was expressed only in the ovary and testis. No DAZL1 product was observed in the heart, liver or brain cDNA. The smaller PCR products in the liver and heart were non-specific products since their presence did not depend on the presence or absence of the reverse transcriptase enzyme.



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Figure 1. (a) Reverse transcription–polymerase chain reaction analysis of DAZL1 expression in cDNA derived from human heart, liver, ovary, testis and brain. The expected product size of the DAZL1 cDNA band is 128 bp. (b) Western blots of DAZ/DAZL1 proteins in human testis. The preimmune antisera for antibody 149 or antibody 150 does not show any specific signals. Antiserum 149 recognizes the DAZL1 protein and a DAZ protein of ~46 kDa (considering the sequence of the cDNA which have been identified in human testis (see Yen et al., 1997), this represents a DAZ protein with nine DAZ repeats; a much less abundant DAZ protein containing ~12–15 DAZ repeats can also be observed with longer exposures). Antiserum 150 recognizes only the DAZL1 protein since the peptide it was raised against is not present in the DAZ protein.

 
Western blotting of DAZ and DAZL1 proteins from human testis
Two peptides, HGKKLKLGPAIRKQKL (peptide 149 which is common to all DAZ family members) and EVDPGADILPNECSVHD (peptide 150 which is identical, except for conservative changes in amino acids shown italicized, in mouse and human DAZL1 homologues but is not present in DAZ protein), were then used to produce polyclonal antibodies. We anticipated that, on Western blots of testis proteins, antiserum 149 would recognize a 33 kDa protein corresponding to DAZL1 and a protein corresponding to DAZ (the predominant DAZ protein species recognized by this antiserum is 46 kDa and would contain ~10 DAZ repeat units). In contrast, antiserum 150 would only recognize the autosomally encoded DAZL1 proteins expressed in testis (Figure 1bGo). Because adult females have fewer germ cells per gonad mass than men (the bulk of the ovary is somatic tissue), we did not necessarily expect to observe a signal corresponding to the protein product in ovaries if the protein localized to germ cells. Indeed, on Western blots, we do see the expected testis-specific bands of the correct molecular weights (competition with the immunizing peptides abolished all of these signals on western blots) but did not observe a corresponding band in ovary, leukocytes, or liver extracts. Thus, we turned to immunohistochemical methods to determine if DAZL1 protein was made in the ovary and, if so, what is the distribution of DAZL1 protein in different cell types and subcellular compartments.

Immunohistochemical localization of DAZL1 protein in fetal and adult ovary
We examined the localization of the DAZL1 protein using antisera 149 and 150 on tissue sections from fetal ovary (19 weeks gestation; at this time nearly all germ cells have entered meiosis) and adult ovary from a 37 year old female (preimmune and antiserum 149) and a 44 year old female (antiserum 150). Results are shown in Figure 2Go. We do not observe any staining of ovarian tissues when treated with preimmune serum (Figure 2a and bGo). In contrast, in both fetal and adult ovarian sections, we see staining with antiserum 149 (Figure 2c and dGo) and antiserum 150 (Figure 2e and fGo). The most-intense signals are observed in the cytoplasm of the oocytes in developing follicles in the fetus and adult (Figures 2c–fGo) but the granulosa cells also appear to stain weakly for DAZL1 protein (Figure 2fGo). This is in close agreement with the distribution of Dazl1 protein in mice (Ruggiu et al., 1997Go). Mouse Dazl1 protein is most abundant in oocytes.



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Figure 2. Immunohistochemical localization of DAZL1 protein in human ovary. The localization of the DAZL1 protein was determined using preimmune antisera (a, b), antiserum 149 which recognizes the conserved RNA binding domain of all DAZ/DAZL1 family members (c, d) and antiserum 150 which recognizes a domain specific to the carboxyterminus of DAZL1 (e, f). Tissue sections are from fetal ovary, 19 weeks gestation (a, c, e) and adult ovary from a 37 year old female (b, d) and a 44 year old female (f). Staining is observed in oogonium, primary and secondary follicles and in granulosa cells. Bar = 10 µm (all panels).

 
Discussion

Dazl1 function in mice
Experiments with Dazl1 null mutants in mice are likely to be most informative regarding the phenotypes that might be associated with loss of function of the human DAZL1 gene (Ruggiu et al., 1997Go). In the mouse, the Dazl1 gene is expressed in both male and female germ cells. Deletion of both chromosomal copies of this gene causes infertility in both sexes before meiosis. Most mice lose their germ cells prenatally before day 15 of development (mouse gestation is 21 days duration). In male mice, there appears to be dosage-sensitivity of the Dazl1 genes, as well. Male mice containing two copies of Dazl1 are normal, those with no copies are sterile, and those with just one copy have lower sperm counts with abnormally shaped germ cells. Although we know that female mice with two functional copies of Dazl1 are fertile, whereas those with no copies are sterile, a determination of the germ cell number in female mice with just one copy of Dazl1 is more difficult and has not yet been done (Ruggiu et al., 1997Go). Taken together, however, these observations from model organisms provide compelling evidence that the DAZ/DAZL1 gene family is required for germ cell development in humans. This is further suggested by the fact that deletions encompassing the DAZ genes on the Y chromosome are associated with infertility in men (Reijo et al., 1995Go, 1996bGo; Pryor et al., 1997Go). However, the role of the DAZL1 gene in determining fertility of women has not yet been explored.

Similar distributions of mouse Dazl1 and human DAZL1 proteins
The results reported here indicate that the DAZL1 protein is most abundant in the cytoplasm of the oocytes in developing follicles in the fetus and adult and that the granulosa cells also appear to contain lesser amounts of DAZL1 protein. This distribution of human DAZL1 is similar to that of mouse Dazl1. The distribution is also similar to that of the regulatory proteins, leptin and STAT3, in that these proteins also localize to both oocytes and follicular cells. In fact, the localization of these proteins to these compartments has been suggested to influence establishment of the axes in fertilized oocytes (Antczak and Blerkom, 1997). However, since there is no evidence that the granulosa cells are defective in mice containing a disruption of the mouse Dazl1 gene or that embryogenesis is defective (Ruggiu et al., 1997Go), we expect that the phenotypes associated with DAZL1 mutations will be restricted to the germ cells only.

We have previously found that in men, the DAZ and DAZL1 proteins were present in both the nucleus and cytoplasm of male gonocytes and were abundant in the nuclei of spermatogonia. Then at meiosis, both proteins relocated to the germ cell cytoplasm (R.Reijo et al., unpublished results). These results, together with findings in diverse species including flies where a similar redistribution occurs, suggest that proteins encoded by the DAZ family may function in multiple cellular compartments and at multiple points in germ cell development in men. They may act during meiosis and much earlier, during the establishment of spermatogonial stem cell populations (R.Reijo et al., unpublished results). In contrast, we have found that the DAZL1 protein in females appears strictly cytoplasmic.

Expected phenotypes associated with DAZL1 mutations in humans
Based on the similarity of the distribution of human DAZL1 protein and mouse Dazl1 protein, we infer that mutations that cause loss of function of the DAZL1 genes would mirror loss of function mutations in mice and probably cause oocyte depletion resulting either in primary amenorrhoea, due perhaps to prenatal loss of the entire oocyte population, or premature ovarian failure, due to depletion of a smaller than average oocyte reserve. Whether a woman presents with primary amenorrhoea or premature ovarian failure is likely to depend on whether one or both copies of the DAZL1 genes are deleted or mutated and on the severity of mutations in the gene. For example, a stop codon introduced before the RNA-binding domain is translated would probably cause a more severe phenotype than an amino acid substitution near the carboxyl terminus of the protein.

The DAZL1 gene and the genetics of primary amenorrhoea and premature ovarian failure
Primary amenorrhoea and premature ovarian failure both may be caused by a depletion of oocyte reserves. Primary amenorrhoea is characterized by the absence of menses by the age of 16 and is most commonly associated with gonadal dysgenesis due to Turner's syndrome, a condition caused by abnormalities in or the absence of one of the X chromosomes. Girls with Turner's syndrome may contain a normal number of primary follicles at birth but usually lose all their ova by puberty (Fox, 1992Go). Besides Turner's syndrome, other causes of primary amenorrhoea are hormonal imbalances or physical abnormalities such as the absence of Fallopian tubes, the uterus, or vagina (reviewed in Yen, 1991). Approximately 25% of instances of primary amenorrhoea may be idiopathic; unidentified genetic causes cannot be ruled out. Mice that carry no functional Dazl1 genes have no germ cells at birth (Ruggiu et al., 1997Go). By comparison, then, we might expect that women who lack a functional copy of the DAZL1 gene may present with idiopathic primary amenorrhoea.

In contrast to primary amenorrhoea, premature ovarian failure has been defined as the advent of secondary amenorrhoea with elevated gonadotrophins occurring prior to 40 years of age and may occur when the store of follicles within the ovary is exhausted (Coulam, 1982Go). Approximately 1% of women encounter premature ovarian failure (Coulam et al., 1986Go). Though premature ovarian failure may result from hormonal abnormalities, tumorigenesis, or environmental factors such as exposure to radiation therapy or chemotherapy, most cases are idiopathic. Familial clustering of ovarian failure has been noted in several studies (for example, see Mattison et al., 1984; Powell et al., 1994; Cramer et al., 1995). In fact, in one study 37.5% of premature ovarian cases reported a family history of premature ovarian failure and the overall risk for premature ovarian failure if a mother or sister had experienced premature ovarian failure was >6-fold greater than when no family history was observed (Cramer et al., 1995Go). Genetic causes of premature ovarian failure have been linked to X chromosome deletions or abnormalities (Powell et al., 1994Go); they have not yet been linked to chromosome 3, especially to 3p24 where the DAZL1 gene maps. Perhaps disruption of a single copy of the DAZL1 gene, or less severe mutations in both copies of the gene, may lead to premature ovarian failure. Women with these milder mutations would form germ cells and may ovulate for some time, but their reproductive life would be shortened due to a lower number of primary follicles being formed prenatally.

Mutation scanning of the DAZL1 genes in infertile women?
Based on the similarity of expression and localization of the human DAZL1 protein and mouse Dazl1 protein, it seems reasonable to consider the DAZL1 gene as a candidate fertility factor in women and it seems appropriate to search for mutations in the DAZL1 gene in peripheral blood DNA from women with either premature ovarian failure or primary amenorrhoea. A combination of karyotypic analysis, deletion analysis and mutational analysis would be suitable. Karyotypic analysis, for example, has been used to identify rearrangements of the X chromosome that cause premature ovarian failure (Powell et al., 1994Go). It similarly could provide information on rearrangements or deletions of the region of chromosome 3 to which the DAZL1 gene maps. Deletion analysis has been used extensively to map fertility loci on the Y chromosome, resulting in the identification of several regions that are required for men to make spermatozoa, including the region that encompasses the DAZ region (Reijo et al., 1995Go; Vogt et al., 1997). The Y chromosome is particularly amenable to deletion analysis of male fertility loci because it is present in just a single copy. However, a modification of deletion analysis referred to as loss of heterozygosity (Strachan and Read, 1996Go) could be used on DNA samples from women who present with primary or secondary ovarian failure to detect small deletions on chromosome 3p24. Finally, in order to examine the DNA of numerous women with primary amenorrhoea or premature ovarian failure for more subtle mutations than karyotypic analysis or deletion analysis can detect, we need a method to screen the DNA rapidly for changes in the coding sequence of the DAZL1 gene before we sequence the gene in individuals who may carry mutations. Two methods of screening DNA from a large number of individuals that are commonly used are heteroduplex analysis and single-stranded conformational polymorphism (SSCP) analysis (Strachan and Read, 1996Go). Both methods can be used to detect stop mutations, small deletions, or hydrophilic/hydrophobic transitions of amino acids in women with ovarian failure.

In summary, the observation in mice that loss of function of the DAZL1 homologue in that organism leads to loss of germ cells in males and females is intriguing (Ruggiu et al., 1997Go). Combined with the demonstration here, using two antisera that recognize human DAZL1 protein, that the human protein is expressed embryonically in germ cells of girls and in the mature oocytes in a manner similar to mice, it suggests that the DAZL1 gene is a candidate fertility factor in women. Since little is known of the genetics of primary amenorrhoea and premature ovarian failure, it seems timely to search for mutations in the DAZL1 gene in peripheral blood DNA from women with primary amenorrhoea or premature ovarian failure in order to begin to understand the genetic basis for such common disorders.


    Acknowledgments
 
This work was supported by a Searle Scholar and Sander Family Foundation Awards to R.A.R.P.


    Notes
 
3 To whom correspondence should be addressed Back


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 Introduction
 Materials and methods
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Submitted on March 1, 1999; accepted on June 24, 1999.