Download The human autosomal gene DAZLA: testis

 1993 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5, No. 12 2013–2017
The human autosomal gene DAZLA: testis specificity
and a candidate for male infertility
Pauline H. Yen*, Ning Ning Chai and Eduardo C. Salido1
Division of Medical Genetics, Harbor-UCLA Medical Center, 1124 W. Carson Street, Torrance, CA 90502-2064,
USA and 1Department of Pathology, Universidad de La Laguna, Spain
Received August 16, 1996; Revised and Accepted October 4, 1996
The DAZ (Deleted in AZoospermia) and DAZLA (DAZlike autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y
chromosome is a strong candidate for the ‘azoospermia factor’ (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion
in a high percentage of males with azoospermia or
severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous
sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse
chromosome 17 and has been considered to be a murine homologue of DAZ. However, the homology
between human DAZ and mouse Dazla is not strong,
and Dazla contains only one of the seven DAZ repeats
found in DAZ. We report the isolation of the human
DAZLA gene by screening a human testis cDNA library
with a DAZ cDNA clone. DAZLA encodes only one DAZ
repeat and shares high homology with the mouse
Dazla, indicating that these two genes are homologues. Using a panel of rodent–human somatic cell
lines and fluorescence in situ hybridization, the DAZLA
gene was mapped to 3p24, a region not known to share
homology with mouse chromosome 17. The DAZLA
gene may be involved in some familial cases of autosomal recessive male infertility.
INTRODUCTION
Mammalian spermatogenesis is a developmental process in
which male germ cells undergo meiosis and complex morphological changes to form mature sperm. Many genes affecting male
fertility have been identified in mice, and they are located both on
autosomes and the sex chromosomes (1,2). The search for human
genes involved in spermatogenesis has been focused on the Y
chromosome long arm, specifically distal Yq11. This region has
been thought to contain an ‘azoospermia factor’ (AZF), because
a high percentage of males with idiopathic azoospermia or severe
oligospermia have deletion of Yq11 sequences (3–10). A recent
*To whom correspondence should be addressed
DDBJ/EMBL/GenBank accession nos U66077, U66078
survey on a large number of infertile men found that deletions can
occur in any one of three non-overlapping regions, indicating that
at least three genes on Yq11 are required for normal spermatogenesis (11). Two candidate genes (or gene families), RBM and
DAZ/SPGY1, have been isolated from Yq11 (8,12,13). Both show
testis specific expression, and both encode proteins with an RNA
binding motif. In addition, RBM encodes four copies of a 37
amino acid (aa) SRGY repeat whereas DAZ encodes seven or 11
copies of a 24 aa DAZ repeat. The functions of these repeats are
unknown. The RBM family consists of 20–40 genes and
pseudogenes scattered over the long arm and the proximal short
arm of the Y chromosome (14). Its mouse homologue Rbm is also
present on the Y chromosome in multiple copies (15,16). There
appears to be more than one copy of the DAZ/SPGY1 gene on
distal Yq11 (8,11; unpublished data). However, DAZ/SPGY1
homologous sequences cannot be detected on the mouse Y
chromosome. Instead, a Dazla (Daz like, autosomal)/Dazh (DAZ
homolog) gene was isolated and mapped to chromosome 17 band
b1 (17,18). The homology between DAZ and Dazla is 72%
identity and 79% similarity at the peptide level, and 83%
similarity in the coding region of the cDNA. In addition, Dazla
contains only one DAZ repeat.
The evidence for DAZ being AZF is only circumstantial. It maps
within the most distal of the three regions that are deleted in some
males with low or no sperm count, but no mutations within the gene
have been identified in infertile males. However, the involvement of
DAZ in spermatogenesis is further supported by a recent discovery
that it shares considerable homology with a Drosophila male
infertility gene, boule (19). The boule gene was identified during
screening for transposon-induced, male sterility mutations in
Drosophila (20). Loss of boule function results in a block of meiotic
division and no sperm production. The boule gene encodes an
RNA-binding motif with 42% identity to that encoded by DAZ/
Dazla. In addition, it contains one copy of the DAZ repeat with 33%
identity to that encoded by DAZ/Dazla. The autosomal localization
of boule and the single copy of the DAZ repeat indicate that boule
is more like Dazla than DAZ.
We report the isolation and characterization of a human DAZLA
on chromosome 3. Sequence comparison among DAZ, DAZLA
and the mouse Dazla showed that Dazla is the mouse homologue
of DAZLA instead of DAZ. The involvement of DAZLA in
families with autosomal recessive male infertility can now be
addressed (21).
2014 Human Molecular Genetics, 1996, Vol. 5, No. 9
Figure 1. DAZ homologous sequences in the human genome. Genomic DNAs
from a normal male (XY) and a female cell line with five X chromosomes (5X)
were digested with EcoRI and hybridized with (A) a DAZ cDNA clone, (B)
DAZLA cDNA clone f7 and (C) a DAZLA cDNA fragment containing 3′ UTR.
RESULTS
The presence of DAZ homologous sequences on human autosomes was indicated by Southern hybridization of a DAZ cDNA
probe to a dosage blot containing DNA from a normal male (XY)
and from a cell line with five X chromosomes (5X) (Fig. 1A).
Weak hybridizing bands were present in the 5X sample, and the
lack of dosage difference of these bands between the XY and 5X
samples indicates that the DAZ homologous sequences are on
autosomes. A human testis cDNA library was therefore screened
with the DAZ cDNA probe, and 15 independent clones were
isolated. Comparison of the restriction maps of the clones with
that of DAZ cDNA showed that seven clones, with inserts ranging
from 1.1 to 3.2 kb, encode a different gene. Hybridization of one
of the clones, f2, to a dosage blot showed autosomal localization
of the gene, which was designated DAZLA (Fig. 1B). DNA
sequence was determined for the entire length of the two longer
clones, f2 and f7, and a few selected regions of the smaller clones.
The 3 kb cDNA includes a 226 bp 5′ untranslated region (UTR),
an open reading frame for a protein of 295 aa residues with an
RNA recognition motif, and a 1.9 kb 3′ UTR (Fig. 2, accession
number U66078). Clone f7 contains a 273 bp insertion after
nucleotide 376, which was shown to represent an unspliced intron
on the basis of PCR amplification of DAZLA genomic clones
using primers flanking the insertion site (data not shown). Clone
f23 contains a smaller insert with a poly A tail attached after
nucleotide 1442, representing the product of alternative polyadenylation. Several single base differences were observed among
the cDNA clones. In the coding region, the T at position 234 in
f2 is replaced by a C in f7 and in three other clones, with a
corresponding amino acid change from isoleucine to threonine.
In the 3′ UTR, f2 and two other clones have three Cs at
nucleotides 2248–2250 whereas f7 has only two Cs at the same
position. At the 3′ end, the nucleotide at 2968 is a C in f2 and in
one other clone, and a G in f7.
Figure 2. Nucleotide and deduced amino acid sequences of the DAZLA cDNA.
The RNA recognition motif (aa 32–117) is in parentheses, the DAZ repeat (aa
167–190) is underlined and the 130 bp segment (nt 832–961) that is unique to
DAZLA is boxed. Sites where an intron interrupts in clone f7 (after nt 376) and
where alternative polyadenylation occurs in clone f23 (after nt 1442) are
indicated with an open and a closed triangle underneath the sequence,
respectively. The three polymorphic sites at positions 234, 2248–2250 and 2968
are double underlined.
The sequences of DAZLA, DAZ and the mouse Dazla were
compared using the GCG gap program. Although the major
transcripts of all three genes are ∼3 kb in length, only 1.4 kb of
Dazla sequence and 1.9 kb of DAZ sequence are available in the
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Figure 3. Sequence comparison of the human DAZ, DAZLA and the mouse
Dazla proteins. The complete sequence of the DAZLA peptide is shown, with the
different amino acid residues in DAZ and Dazla shown above and below,
respectively. The RRM is in parentheses and the consensus RNP-1 and RNP-2 are
underlined. The DAZ repeat is boxed. The segment in DAZ protein that encodes
an extra six DAZ repeats is excluded for comparison and is not shown here.
database. We sequenced one of the DAZ cDNA clones that we
isolated to obtain the remaining sequence of the DAZ transcripts
(accession no. U66077). As for Dazla, comparison is limited to
the available sequence which includes only 200 bp of 3′ UTR.
The overall structure of DAZLA and its predicted protein product
is more similar to that of Dazla than DAZ. Both DAZLA and Dazla
proteins contain only one copy of the DAZ repeat (nt 725–796),
in contrast to the DAZ proteins which contain seven or more DAZ
repeats (8,11; unpublished data). In addition, both DAZLA and
Dazla contain a 130 bp segment (nt 832–961) at the 3′ portion of
the coding region that is absent in DAZ. The peptide sequences are
86% identical and 91% similar between DAZLA and Dazla, as
compared with 78% identical and 83% similar between DAZ and
Dazla, excluding the extra DAZ repeats in DAZ (Fig. 3).
Therefore DAZLA is the human homologue of Dazla. At the DNA
level, DAZLA and Dazla show high similarity only in the coding
region (89%), whereas the 5′ UTR (48% similarity) and 3′ UTR
(75% similarity) are much less conserved. On the other hand,
DAZLA and DAZ share high homology throughout the entire
length, excluding the two segments not shared by the two genes.
These are the 432 bp segment in DAZ that encodes six extra DAZ
repeats and the 130 bp segment in DAZLA mentioned above.
Sequence similarity for the 5′ UTR, coding region and the 3′ UTR
are 81, 90 and 88%, respectively.
The expression of DAZLA was studied using both northern blot
and RT–PCR. For northern analysis a cDNA fragment from the 3′
UTR of DAZLA was used as a probe to minimize hybridization
with DAZ transcripts. To test the specificity of this probe, Southern
hybridization with a dosage blot was performed. Under stringent
conditions the probe detected a strong 4.3 kb autosomal fragment
and a faint 4.5 kb fragment on the Y chromosome (Fig. 1C). On
three northern blots that included poly A+ RNA from 17 human
tissues (see Material and Methods), this probe detected a major 3.5
kb transcript in testis only (Fig. 4A). Transcripts of smaller size
were also detected, however, they were present at a much lower
level. No signal was detected in ovary, indicating that DAZLA is not
expressed there, or only at a very low level (data not shown).
Despite the limitation that the probe also detected DAZ transcripts
to some extent, the absence of hybridization signals in other tissues
Figure 4. Testis specific expression of the DAZLA gene. (A) Hybridization of
a multiple tissue northern blot with a DAZLA cDNA fragment containing 3′
UTR. (B) RT–PCR of testicular RNA using a primer set specific for the DAZLA
transcripts. Samples hT10 and hT2 are from testicular biopsies with normal
spermatogenesis, whereas hT3 and hT4 have Sertoli cells only. Clones f2 and
e11 are DAZLA and DAZ cDNA clones, respectively. In the H2O lane, no cDNA
was added in the PCR reaction.
supports the conclusion that the expression of DAZLA is testis
specific. In our screening of the testis cDNA library, about equal
numbers of DAZ and DAZLA cDNA clones were isolated,
indicating that DAZ and DAZLA are expressed at comparable
levels in testis. The expression of DAZLA in male germ cells was
studied by RT–PCR using RNA samples from testicular biopsies
of normal and infertile males (22; Fig. 4B). Specific amplification
of the DAZLA transcripts was achieved by selecting one of the
primer sequences from the 130 bp segment that is not shared by the
DAZ gene. The primer set spans several introns, and a 4 kb
fragment would occur for genomic DNA amplification (data not
shown). The primer set amplified a 467 bp fragment from DAZLA
cDNA clone f2, but not from DAZ cDNA clone e11. The same
PCR products are present in two samples from males with normal
spermatogenesis (hT10 and hT2), but not in two samples from
males with Sertoli cells only (hT3 and hT4). The testicular cDNA
samples were used for the amplification of a ubiquitously
expressed gene, MIC2, to ensure that adequate cDNA was present
in the samples (23). All the samples amplified a predicted 360 bp
product (data not shown). These results are consistent with germ
cell expression of the DAZLA genes. However, the possibility that
the DAZLA gene is expressed in Sertoli cells only in the presence
of normal germ cells cannot be ruled out.
The DAZLA gene was mapped initially to human chromosome
3 by Southern hybridization of a DAZLA cDNA probe to a panel
of 15 rodent–human somatic cell hybrids containing various
combinations of human chromosomes (24; data not shown). To
fine map the gene on chromosome 3, several genomic clones
were isolated after screening a human genomic library with
DAZLA cDNA. One clone λg12, which was shown to encode the
DAZLA gene by sequencing some of the exons, was used as a
probe in fluorescence in situ hybridization (FISH) mapping on
metaphase human chromosomes. A strong hybridization signal
was observed at 3p24, confirming chromosome 3 localization of
the gene (Fig. 5). No consistent hybridization signal was observed
in any other region of the genome.
2016 Human Molecular Genetics, 1996, Vol. 5, No. 9
Because the mouse Dazla is located on chromosome 17 band
b1, it was predicted that the human DAZLA would be on the short
arm of human chromosome 6, based on homology between the
mouse and human gene maps (25). However, by a somatic cell
mapping panel and FISH, DAZLA was mapped to human
chromosome 3p24, a region not known to contain sequences
homologous to mouse chromosome 17 (26). It is likely that 3p24
represents a new syntenic region for mouse chromosome 17.
However, the possibility that there are additional DAZ-like genes
in the human genome, although unlikely, cannot be ruled out.
Southern hybridization of female DNA with a DAZ cDNA probe
detected more than one fragment (Fig. 1A). Further studies,
characterizing DAZLA genomic clones, will determine whether
all of the hybridizing fragments belong to the DAZLA gene on
chromosome 3.
Figure 5. FISH mapping of the DAZLA gene on human metaphase chromosomes. (a) A portion of a metaphase chromosome spread showing hybridization of a DAZLA genomic clone to chromosome 3p24. (b) The same portion
stained with DAPI to identify chromosome 3.
DISCUSSION
Male infertility is a common problem, affecting 1–3% of couples
(14). Although it can be caused by many different factors,
genetics undoubtedly plays a role in this disorder. The formation
of a mature sperm is a very complex process involving many
genes. Defects in any one of these genes can result in impaired
sperm or no sperm at all. Among infertile males with azoospermia
or severe oligospermia, ∼5–10% have a deletion of the AZF locus
on Yq11. These deletions most likely represent new mutations,
since defects on the Y chromosome that severely affect the
fertility of an individual are not likely to be transmitted. In a rare
case where the father has the same deletion as a severely
oligospermic son, it is likely that one of the father’s sperm,
although greatly reduced in number, was able to fertilize an egg
(11). Defects in autosomal genes required for spermatogenesis
will be inherited in a male-limited autosomal fashion. Families
with multiple infertile males have been reported (21). In a
case-control study to determine whether infertility in men is
familial, it was concluded that infertility may be inherited in an
autosomal recessive mode for over half of the cases. No linkage
study to map the male infertility gene has been reported so far.
The DAZLA gene we have isolated is a definite candidate gene for
autosomal recessive male infertility. The mapping of DAZLA to
3p24–25 will permit linkage studies using polymorphic markers
from this region. In addition, we have identified three polymorphisms within the DAZLA gene. In fact among the DAZLA cDNA
clones isolated, we have already identified three haplotypes. They
are C3C in f1, T3C in f2 and C2G in f7, in which the first letter
denotes the base at position 234, the middle number denotes the
number of Cs at positions 2248–2250, and the last letter denotes
the base at position 2968. (Note that the testis cDNA library used
was constructed from RNAs pooled from four males.) The
putative polymorphisms within the DAZLA gene may be useful
for linkage studies, although we do not yet know their frequencies
in the general population.
MATERIALS AND METHODS
The human testis cDNA library (939202) was purchased from
Stratagene (La Jolla, CA), whereas the human genomic library
(HL1067J) was purchased from Clontech (Palo Alto, CA). The
libraries were plated out at a density of 3 × 104 p.f.u. per 100 mm
plate and screened with a DAZ or DAZLA cDNA probe according
to standard protocols (27).
Three human multiple tissue northern blots were purchased from
Clontech. Blot no. 7760-1 contains poly A+ RNAs from heart, brain,
placenta, lung, liver, skeletal muscle, kidney and pancreas and blot
no. 7766-1 contains RNAs from spleen, thymus, prostate, testis,
uterus, small intestine, colon (mucosal lining) and peripheral blood
leukocytes. Blot no. 7759-1 is similar to blot 7766-1 except that it
contains ovary RNA instead of uterus RNA. Hybridization was
carried out in ExpressHyb Hybridization Solution (#8015, Clontech)
at 68C for 1 h according to the manufacturer’s protocol. The probe
was a 772 bp HindIII–EcoRI fragment (nt 1837–2608) from the 3′
UTR of the DAZLA cDNA clone.
Purification of RNA from testicular samples and RT-PCR were
carried out as described previously (22). The PCR primers were
PRDAZ28: 5′-ATGACGTGGATGTGCAG (nt 492–508) and
PRDAZ29: 5′-TTGTGGGCCATTTCCAG (nt 958–942). Reactions were carried out at 94C for 1 min, 52C for 1 min and 72C
for 1 min for 30 cycles with a final extension at 72C for 10 min.
The products were separated on a 3% Nusieve-agarose (3:1) gel.
FISH mapping of the DAZLA gene was carried out using the
services of SeeDNA Biotech Inc. (North York, Ontario, Canada).
DAZLA genomic clone λDAZLA-g12 with an insert of 15 kb,
was used as a probe for FISH studies. Slides preparation, in situ
hybridization and signal detection were carried out according to
Heng et al. (28). Regional assignment was based on superimposing FISH signals with DAPI banded chromosomes. Ten photographs were analyzed.
ACKNOWLEDGEMENTS
We thank D. Page for the DAZ cDNA clone, Ann Chandley,
Henry Lin and T. K. Mohandas for helpful discussion, and
Hsiao-fen Huang for excellent technical assistance. The work was
supported by grant HD28009 from the National Institutes of
Health.
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