daz-1, the single DAZ homolog in the nematode Caenorhabditis elegans has been identified and characterized. Loss of daz-1 function causes sterility in hermaphrodites, by blocking oogenesis at the pachytene stage of meiosis I. Epistasis analysis suggests that this gene executes its function after gld-1, which governs the early pachytene stage in the oogenic pathway. Spermatogenesis does not appear to be affected in daz-1 hermaphrodites. Males defective in daz-1 produced sperm fully competent in fertilization. Analysis employing sex-determination mutants indicates that the daz-1 function is required for meiosis of female germline regardless of the sex of the soma. Transcription of daz-1 is restricted to the germline, starting prior to the onset of meiosis and is most conspicuous in cells undergoing oogenesis. Thus, daz-1 in C. elegans is an essential factor for female meiosis but, unlike other DAZ family members so far reported, it is dispensable for male meiosis (Karashima, 2000).
DAZ family genes are conserved from nematode to mammals. However, no DAZ homolog has been found in unicellular organisms; this includes meiosis-proficient eukaryotic microbes like budding and fission yeast as well as bacteria. This may imply that the emergence of the DAZ family gene in the course of evolution was associated with the increase of complexity in the mechanisms of meiosis in multicellular organisms. All of the DAZ family genes so far investigated are expressed exclusively in the germline, but their sex-specificity is not identical. The DAZ family genes in Drosophila and C. elegans are required for gametogenesis in one sex: Drosophila boule for spermatogenesis (Eberhart, 1996) and C. elegans daz-1 for oogenesis. Murine Dazla is required for both oogenesis and spermatogenesis, but differently: in Dazla-deficient mice, oogenesis appears to be normal until it is arrested at the pachytene stage, hence mimicking the situation in C. elegans, whereas the male germline generates fewer germ cells, which do not proceed beyond the spermatogonial stage. It is unknown whether the human counterpart of murine Dazla, namely DAZLA/DAZH, is involved in gametogenesis. A deletion of the human DAZ cluster on Y chromosome results in a wide range of spermatogenic deficiency. This variability may reflect the functional redundancy and divergence between DAZ and DAZLA/DAZH (Karashima, 2000).
How have the DAZ family genes acquired such versatile sex-specificity during evolution? Yeast has only one mode of meiosis, i.e., diploid cells generate four spores. In contrast, higher organisms produce asymmetrical gametes. To regulate spermatogenesis and oogenesis independently, each program would require a different mode of meiosis: these two modes should be at least partly distinct from one another. Thus, it is assumed that multicellular organisms have developed two types of mechanisms for meiotic regulation, one of which uses the DAZ family gene at the pachytene stage and the other which does not. Meanwhile, strategies for sex determination are strikingly divergent among species, with no overlapping molecular mechanisms being discovered in C. elegans, Drosophila and mammals. Taken together, during the evolution from a primitive multicellular organism, some organisms may have employed the DAZ-dependent meiosis for spermatogenesis and the DAZ-independent meiosis for oogenesis, whereas others did vice versa. Hopefully more extensive analysis of the DAZ family genes and more intensive analysis of their molecular function will prove or disprove the validity of this hypothesis (Karashima, 2000).
The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. Mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, it is proposed that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis (Maruyama, 2005).
Based on the sequence similarity of the RNA-recognition motifs, the DAZ family has been divided into two subgroups, namely, BOULE and DAZL. It was inferred that the BOULE group is the more ancient, that the DAZL group derived from BOULE by duplication in the ancestor of vertebrates, and that DAZ, which belongs to the DAZL group, was generated in primates by duplication of DAZL. The BOULE members other than C. elegans DAZ-1, i.e., Drosophila boule and murine and human BOULE, are expressed in testis but neither ovary nor primordial germ cells. In contrast, all the DAZL members appear to be expressed in both male and female germlines since the primordial germ cells stage. C. elegans DAZ-1 is undetectable in germ cell precursors in the embryo but emerges in both male and female germ cells as they start proliferation at the early larval stage. This expression profile is rather similar to that of the DAZL subgroup. In addition, the function of C. elegans DAZ-1 in vivo is apparently female specific, contrasting with the male specificity of Drosophila Boule. Thus, the expression profiles and the loss-of-function phenotypes of the DAZ family members do not necessarily correlate with the subgrouping that relies on sequence similarity. This divergence of the in vivo function even within the BOULE subgroup implies that the role of the DAZ family members might have been subjected to rapid modification in the course of evolution. However, because some DAZ members can substitute for others in different species, at least partially, they appear to maintain a common biochemical function, presumably as translational regulators utilizing their conserved RNA-binding activity. Further characterization of DAZ family members, including identification of the downstream target genes, is awaited to understand how this family controls germline development and how it has evolved (Maruyama, 2005).
In many species, DAZ homologous genes encode RNA-binding proteins containing two conserved motifs, namely the RNA-recognition motif (RRM) and the DAZ motif. Genetic analysis and gene disruption studies have demonstrated that DAZ family proteins play important roles in gametogenesis. However, little is known about the biochemical functions of DAZ family proteins. Using in vitro selection and UV-crosslinking experiments, the sequence 'GUUC' was identified as the target RNA sequence of zebrafish DAZ-like protein (zDAZL). In transfection experiments, zDAZL protein activates translation in a manner dependent on the binding sequence in the 3'UTR of the Drosophila twine gene or zDazl gene. Moreover, it is highly likely that the zDAZL protein associates with polysomes through the DAZ motif in vivo, and that the association with polysomes is indispensable for translational activation. This is the first report that the DAZ family protein directly promotes the translation of the target mRNAs in vertebrates. This study provides important insights into the molecular mechanisms underlying the post-transcriptional regulation of DAZ family proteins in gametogenesis (Maegawa, 2002).
A localized RNA component of Xenopus germ plasm has been identified. This RNA, Xdazl (Xenopus DAZ-like), encodes a protein homologous to human DAZ (Deleted in Azoospermia), vertebrate DAZL and Drosophila Boule proteins. Similarity among the DAZ homologs is high: Xdazl and DAZ have 42% identical residues overall, and Xdazl shares approximately 60% identical residues with mouse and human Dazl proteins. Similarity to the Boule protein is less, with 27% of the residues remaining conserved from flies to frogs. Xdazl also contains some residues corresponding to the 24 amino acid DAZ repeat, although the Xdazl protein has a three amino acid insertion, AIQ, in the middle of the motif. In general, the proteins appear well conserved within the RNP domain and more divergent at the amino terminus and in the C-terminal domain. At the nucleotide level, Xdazl is most closely related to the murine Dazl gene. In a stretch of 800 nucleotides constituting most of the coding region, the two genes are approximately 70% identical overall. Also, the sequences of many inferred exon boundaries are highly conserved from frog to man. The high amino acid and nucleotide sequence conservation among the DAZ members suggests that the genes have evolved from a common ancestor and may be involved in similar functions in all organisms (Houston, 1998).
Xdazl RNA is detected in the mitochondrial cloud and vegetal cortex of oocytes. In early embryos, the RNA is localized exclusively in the germ plasm. Consistent with other organisms, Xdazl RNA is also expressed in the spermatogonia and spermatocytes of frog testis. To determine if the RNA is present during specific stages of spermatogenesis, sections of adult and juvenile testis tissue were hybridized with DIG-labeled antisense RNA probes. In both the adult (and juvenile), Xdazl RNA is found in discrete spermatocysts. The majority of Xdazl-expressing cells have been identified as spermatocytes. The observation of metaphase plates in some of the spermatocyte cysts confirms that Xdazl RNA is expressed in the frog testis in the cells undergoing meiotic cell division. Some staining is also detected in spermatogonia; however, Xdazl RNA is consistently absent in spermatids and mature sperm. Proteins in the DAZ-family contain a conserved RNP domain, implying an RNA-binding function. Xdazl can function in vitro as an RNA-binding protein. To determine if the function of Xdazl in spermatogenesis is conserved, the Xdazl cDNA was introduced into boule flies. This resulted in rescue of the boule meiotic entry phenotype, including formation of spindles, phosphorylation of histone H3 and completion of meiotic cell division. Overall, these results suggest that Xdazl may be important for primordial germ cell specification in the early embryo and may play a role analogous to Boule in promoting meiotic cell division (Houston, 1998).
Germ plasm is morphologically similar in all organisms where it is found and is typically composed of a fibrillar 'germinal cytoplasm', electron-dense germinal granules, mitochondria and ribosomes. In Xenopus, the germ plasm is present in eggs as numerous discrete islands at the vegetal pole. These islands aggregate after fertilization, a process that requires a kinesin-like protein, Xklp-1. Germ plasm is segregated unequally during cleavage stages until gastrulation, at which time the germ plasm becomes perinuclear and is divided equally among daughter cells. During subsequent embryogenesis, primorial germ cells (PGCs), carrying the germ plasm, remain in the endoderm and are thought to undergo 2-3 cell divisions. Around stage 32/33 (late tailbud stage), the PGCs begin to migrate dorsally through the lateral endoderm. By early tadpole (stage 40), the PGCs accumulate in the dorsal crest of the posterior endoderm and are subsequently incorporated into the lateral plate mesoderm that forms the dorsal mesentery. In later stages, PGC migration continues to the dorsal body wall and then laterally to the forming genital ridges (Houston, 2000).
In Xenopus, several RNAs have been found localized to the germ plasm but none of these have previously been tested for roles in PGC specification. Several of these RNAs are similar to important Drosophila pole plasm components; Xcat2 encodes a Nanos homolog and Xlsirts encodes an untranslated RNA. Xdazl is expressed in the mitochondrial cloud of stage I oocytes, the source of germ plasm material, and remains expressed in the germ plasm until the neurula stage. Xdazl RNA is also abundantly expressed in germ cells of the testis, but not in any of the somatic tissues. Xdazl encodes an RNA-binding protein and is highly related to genes of the Deleted in Azoospermia (DAZ) (Houston, 2000 and references therein).
Focused upon was the question of whether depletion of maternal Xdazl could cause a deficiency in histologically identifiable PGCs in tadpoles. Xdaz-1 was depleted from oocytes by Xdaz-1 antisense mRNA. Xdazl-depleted oocytes were fertilized and the embryos were raised to the tadpole stage (stage 43/44). PGC numbers in Xdazl-depleted embryos are greatly reduced. In many cases, PGCs are completely eliminated. It was asked whether there were PGCs present in the stage 40 embryos that lacked Xpat (a PGC-specific molecular marker) RNA. Xdazl-depleted stage 40 embryos were shown to lack Xpat RNA and cells with the morphological features of PGCs could not be found. This observation suggests that PGC migration does not occur in the absence of Xpat RNA. Additionally, the lack of Xpat-containing cells remaining in the gut indicates that PGC migration is not merely delayed and that residual PGCs do not persist in the endoderm of Xdazl-depleted embryos (Houston, 2000).
How germ cell specification occurs remains a fundamental question in embryogenesis. The embryos of several model organisms contain germ cell determinants (germ plasm) that segregate to germ cell precursors. In other animals, including mice, germ cells form in response to regulative mechanisms during development. To investigate germ cell determination in urodeles, where germ plasm has never been conclusively identified, DAZ-like sequence was cloned from axolotls and termed Axdazl. Axdazl is homologous to Xdazl, a component of Xenopus germ plasm found in the vegetal pole of oocytes and eggs. Axdazl RNA is not localized in axolotl oocytes, and, furthermore, these oocytes do not contain the mitochondrial cloud that localizes Xdazl and other germ plasm components in Xenopus. Maternal Axdazl RNA is inherited in the animal cap and equatorial region of early embryos. At gastrula, neurula, and tailbud stages, Axdazl RNA is widely distributed. Axdazl first shows cell-specific expression in primordial germ cells (PGCs) approaching the gonad at stage 40, when nuage (germ plasm) appears in PGCs. These results suggest that, in axolotls, germ plasm components are insufficient to specify germ cells (Johnson, 2001).
Deletions of portions of the Y chromosome long arm have been detected in 12 of 89 men with azoospermia (no sperm production). No Y deletions were detected in their male relatives or in 90 other fertile males. The 12 deletions overlap, defining a region likely to contain one or more genes required for spermatogenesis (the Azoospermia Factor, AZF). Deletion of the AZF region is associated with highly variable testicular defects, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids. No evidence has been found of YRRM genes, recently proposed as AZF candidates, in the AZF region. The region contains a single-copy gene, DAZ (Deleted in AZoospermia), which is transcribed in the adult testis and appears to encode an RNA binding protein (Reijo, 1995).
Deletion of the Azoospermia Factor (AZF) region of the human Y chromosome results in spermatogenic failure. While the identity of the critical missing gene has yet to be established, a strong candidate is the putative RNA-binding protein DAZ (Deleted in Azoospermia). The mouse homolog of DAZ is described. Unlike human DAZ, which is Y-linked, in mouse the Dazh (DAZ homolog) gene maps to chromosome 17. Nonetheless, the predicted amino acid sequences of the gene products are quite similar, especially in their RNP/RRM (putative RNA-binding) domains, and both genes are transcribed predominantly in the testis; the mouse gene is transcribed at a lower level in ovaries. Dazh transcripts were not detected in testes of mice that lack germ cells. In testes of wildtype mice, Dazh transcription is detectable 1 day after birth (when the only germ cells are prospermatogonia), increases steadily as spermatogonial stem cells appear, plateaus as the first wave of spermatogenic cells enters meiosis (10 days after birth), and is sustained at this level thereafter. This unique pattern of expression suggests that Dazh participates in differentiation, proliferation, or maintenance of germ cell founder populations before, during, and after the pubertal onset of spermatogenesis. Such functions could readily account for the diverse spermatogenic defects observed in human males with AZF deletions (Reijo, 1996).
It is widely believed that most or all Y-chromosomal genes were once shared with the X chromosome. The DAZ gene is a candidate for the human Y-chromosomal Azoospermia Factor (AZF). Multiple copies of DAZ (> 99% identical in DNA sequence) clustered in the AZF region and a functional DAZ homolog log (DAZH) on human chromosome 3 are reported. The entire gene family appears to be expressed in germ cells. Sequence analysis indicates that the Y-chromosomal DAZ cluster arose during primate evolution by (1) transposing the autosomal gene to the Y; (2) amplifying and pruning exons within the transposed gene and (3) amplifying the modified gene. These results challenge prevailing views of sex chromosome evolution, suggesting that acquisition of autosomal fertility genes is an important process in Y chromosome evolution. (Saxena, 1996)
To understand the DAZ gene family and its expression, the DAZ genomic structure and RNA transcripts in numerous males, as well as several DAZ cDNA clones have been analyzed. The results of genomic Southern blot have shown that each male contains multiple DAZ genes with varying numbers of DAZ repeats, and that the copy number of the DAZ repeats are polymorphic in the population. The presence of multiple species of DAZ transcripts with different copy number and the arrangement of the DAZ repeats in an individual suggest that more than one DAZ gene is transcribed. The existence of multiple functional DAZ genes complicates the analysis of genotype/phenotype correlations among males with varying sperm counts (Yen, 1997).
The recent transposition to the Y chromosome of the autosomal DAZL1 gene, potentially involved in germ cell development, has created a unique opportunity to study the rate of Y chromosome evolution and assess the selective forces that may act upon such genes, and provides a new estimate of the male-to-female mutation rate (alpham). Two different Y-located DAZ sequences have been observed in all Old World monkeys, apes and humans. Different DAZ copies originate from independent amplification events in each primate lineage. A comparison of autosomal DAZL1 and Y-linked DAZ intron sequences gives a new figure for male-to-female mutation rates of alpham = 4. It was found that human DAZ exons and introns are evolving at the same rate, implying neutral genetic drift and the absence of any functional selective pressures. It is therefore hypothesized that Y-linked DAZ plays little, or a limited, role in human spermatogenesis. The two copies of DAZ in man appear to be due to a relatively recent duplication event (55,000 to 200,000 years). A worldwide survey of 67 men from five continents representing 19 distinct populations has showen that most males have both DAZ variants. This implies a common origin for the Y chromosome consistent with a recent 'out of Africa' origin of the human race (Agulnik, 1998).
Defects in human germ cell development are common and yet little is known of genes required for germ cell development in men and women. The pathways that develop germ cells appear to be conserved broadly, at least in outline, in organisms as diverse as flies and humans beginning with allocation of cells to the germ cell lineage, migration of these cells to the fetal gonad, mitotic proliferation and meiosis of the germ cells, and maturation into sperm and eggs. In model organisms, a few thousand genes may be required for germ cell development including meiosis. To date, however, no genes that regulate critical steps of reproduction have been shown to be functionally conserved from flies to humans. This may be due in part to strong selective pressures that are thought to drive reproductive genes to high degrees of divergence. This study investigated the micro- and macro-evolution of the Boule gene, a member of the human DAZ gene family, within primates, within mammals and within metazoans. Sequence divergence of Boule is unexpectedly low and rapid evolution is not detectable. The evolutionary analysis of Boule has been extended to the level of phyla and a human Boule transgene can advance meiosis in infertile boule mutant flies. This is the first demonstration that a human reproductive gene can rescue reproductive defects in a fly. These studies lend strong support to the idea that Boule may encode a key conserved switch that regulates progression of germ cells through meiosis in men (Xu, 2003).
The DAZ (Deleted in AZoospermia)and DAZLA (DAZ-like 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 homolog 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. The human DAZLA gene has been isolated 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 homologs. 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 (Yen, 1996).
A homolog of the human Y linked DAZ gene has been isolated from mouse. This gene, Dazla (Daz like, autosomal), is autosomal, located on chromosome 17 and apparently single copy. The predicted protein is highly homologous to that encoded by the DAZ gene in the N-terminal regions of the two proteins and this homology is not confined to the RNA binding domain. Analysis of its expression pattern by reverse transcription PCR shows that the transcript is only detectable in male and female gonads and that testes lacking germ cells do not contain detectable amounts of transcript. A Y-linked DAZ homolog could not be detected in mouse and these results point to the possibility of a role for autosomal RNA binding proteins in mammalian gametogenesis (Cooke, 1996).
A series of human testis poly(A) cDNA clones has been isolated by cross-hybridization to SPGY1, a Y gene homologous to DAZ. Their sequence analysis reveals an identical nucleotide composition in different 'full-length' clones, suggesting that all were encoded by the same gene. This gene has been mapped to the short arm of chromosome 3 and has been designated SPGYLA (SPGY like autosomal). Comparison of the SPGYLA cDNA sequence with the cDNA sequences of DAZ and SPGY1 has revealed two prominent differences. The tandem repetitive structure of 72 bp sequence units (DAZ repeats) is absent. SPGYLA contains only one 72 bp sequence unit. Downstream of it, a specific 130 bp sequence domain is present that is absent in DAZ and SPGY1 but present in the mouse gene Dazla and in the Drosophila gene boule. SPGYLA encodes an RNA binding protein expressed only in the human male gonad. The data presented give strong evidence that not DAZ but SPGYLA is the functional human homolog of Dazla and boule (Shan, 1996).
The DAZLA (DAZ Like Autosomal) gene on human chromosome 3 shares a high degree of homology with the DAZ (Deleted in AZoospermia) gene family on the Y chromosome, a gene family frequently deleted in males with azoospermia or severe oligospermia. The involvement of both DAZ and DAZLA in spermatogenesis is suggested by their testis-specific expression and their homology with a Drosophila male infertility gene, boule. Whereas male infertility resulting from deletion of the DAZ genes on the Y chromosome occurs sporadically, that due to a defective DAZLA gene is expected to be inheritable. The fraction of males with idiopathic azoospermia or oligospermia that harbor mutations in the DAZLA gene remains unknown. As a prerequisite for mutation screening, the genomic structure of the DAZLA gene was elucidated and found to consist of 11 exons spanning 19 kh. The exon/intron boundaries are conserved between DAZ and DAZLA. The 5' end of both genes are hypomethylated in spermatozoa but not in leukocytes or placenta, consistent with the expression pattern of the genes. The genomic structure of DAZLA paves the way for mutation detection in families with autosomal recessive male infertility (Chai, 1997).
RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, because they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homolog is essential for spermatogenesis in Drosophila. Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells (Ruggiu, 1997).
The human homolog of the mouse germ cell-specific transcript Tpx2, which maps to mouse chromosome 17, has been isolated. Sequence analysis shows that the human gene is part of the DAZ (Deleted in Azoospermia) family, represents the human homolog of the mouse Dazla and Drosophila boule genes, and is termed DAZLA. Like Dazla and boule, DAZLA is single copy and maps to 3p25. This defines a new region of synteny between mouse chromosome 17 and human chromosome 3. Unlike DAZ, which has multiple DAZ repeats, DAZLA encodes a putative RNA-binding protein with a single RNA-binding motif and a single DAZ repeat. DAZLA is more closely related to Dazla in the mouse than to the Y-linked homolog DAZ (88% identity overall with mouse Dazla compared to 76% identity with the human DAZ protein sequence). Southern blot analysis showed that DAZLA is autosomal in all mammals tested and that DAZ has been recently translocated to the Y chromosome, sometime after the divergence of Old World and New World primates. To investigate the evolutionary relatedness of DAZLA and DAZ further, their partial genomic structures were obtained and compared. This has revealed that the genomic organization of both genes in the 5' region is highly conserved. DAZLA is a new member of the DAZ family of genes, which is associated with spermatogenesis and male sterility. Familial cases of male infertility in humans show an autosomal recessive mode of inheritance. It is possible that some of these families may carry mutations in the DAZLA gene (Seboun, 1997).
The DAZ gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. DAZL1 and DAZ bind similarly to various RNA homopolymers in vitro. An antibody against the human DAZL1 was used to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. These results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation (Tsui, 2000).
DAZ is an RNA-binding protein encoded by a region on the Y chromosome implicated in infertility, and DAZ-like (Dazl) proteins are master regulators of germ line gene expression in all animals. In mice Dazl is expressed only in germ cells and is necessary for meiosis. A dual approach was taken to understand the RNA-binding specificity of the Dazl protein: (1) traditional SELEX and (2) a novel tri-hybrid screen. Both approaches led to the same conclusion, namely that Dazl binds oligo(U) stretches interspersed by G or C residues. In a directed tri-hybrid assay the strongest interaction was with the consensus (GUn)n. This motif is found in the 5' UTR of CDC25C whose homologue is thought to be the target of Boule, the Dazl homologue in flies. CDC25C 5' UTR also interacts specifically with Dazl in vitro. The tri-hybrid screen retrieved UTRs of known genes that may be physiological substrates of Dazl (Venables, 2001).
Members of the Pumilio and DAZL family of RNA binding proteins are required for germ cell development in Drosophila, Xenopus, and Caenorhabditis elegans. This study reports identification and characterization of RNA sequences to which PUM2 and DAZL bind. Human PUM2 specifically recognizes the Drosophila Pumilio RNA target (the NRE or Nanos regulator element sequence); single nucleotide changes in the NRE abolished PUM2 binding. Then, coimmunoprecipitation was used to isolate human transcripts specifically bound by PUM2 and DAZL and subsequently those were identified that contain NRE-like sequence elements. The interacting proteins, PUM2 and DAZL, are capable of binding the same RNA target and mRNA sequences bound by both proteins in the 3'UTR of human SDAD1 mRNA were further characterized. Taken together, the results define sequences to which these germ cell-specific RNA binding proteins may bind to promote germ cell development (Fox, 2005).
Germ cell development is complex; it encompasses specification of germ cell fate, mitotic replication of early germ cell populations, and meiotic and postmeiotic development. Meiosis alone may require several hundred genes, including homologs of the BOULE (BOL) and PUMILIO (PUM) gene families. Both BOL and PUM homologs encode germ cell specific RNA binding proteins in diverse organisms where they are required for germ cell development. Human BOL forms homodimers and is able to interact with a PUMILIO homolog, PUM2. The domain of BOL that is required for dimerization and for interaction with PUM2 was mapped. BOL and PUM2 can form a complex on a subset of PUM2 RNA targets that is distinct from targets bound by PUM2 and another deleted in azoospermia (DAZ) family member, DAZ-like (DAZL). This suggests that RNA sequences bound by PUM2 may be determined by protein interactions. This data also suggests that although the BOL, DAZ, and DAZL proteins are all members of the same gene family, they may function in distinct molecular complexes during human germ cell development (Urano, 2005).
It has been reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, this study reports that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established (Reijo, 2000).
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