Differential conservation and divergence of fertility genes boule and dazl in the rainbow trout

Background

The genes boule and dazl are members of the DAZ (Deleted in Azoospermia) family encoding RNA binding proteins essential for germ cell development. Although dazl exhibits bisexual expression in mitotic and meiotic germ cells in diverse animals, boule shows unisexual meiotic expression in invertebrates and mammals but a bisexual mitotic and meiotic expression in medaka. How boule and dazl have evolved different expression patterns in diverse organisms has remained unknown.

Methodology and Principal Findings

Here we chose the fish rainbow trout (Oncorhynchus mykiss) as a second lower vertebrate model to investigate the expression of boule and dazl. By molecular cloning and sequence comparison, we identified cDNAs encoding the trout Boule and Dazl proteins, which have a conserved RNA-recognition motif and a maximal similarity to their homologs. By RT-PCR analysis, adult RNA expression of trout boule and dazl is restricted to the gonads of both sexes. By chromogenic and two-color fluorescence in situ hybridization, we revealed bisexual and germline-specific expression of boule and dazl. We found that dazl displays conserved expression throughout gametogenesis and concentrates in the Balbinani''s body of early oocytes and the chromatoid body of sperm. Surprisingly, boule exhibits mitotic and meiotic expression in the male but meiosis-specific expression in the female.

Conclusions

Our data underscores differential conservation and divergence of DAZ family genes during vertebrate evolution. We propose a model in which the diversity of boule expression in sex and stage specificity might have resulted from selective loss or gain of its expression in one sex and mitotic germ cells.     

Differential expression of boule and dazl in adult germ cells of the Asian seabass

Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish.     

Boule Is Present in Fish and Bisexually Expressed in Adult and Embryonic Germ Cells of Medaka

Background

The DAZ family genes boule, daz and dazl encode RNA binding proteins essential for fertility of diverse animals including human. dazl has bisexual expression in both mitotic and meiotic germ cells, whereas daz has male premeiotic expression, and boule is largely a unisexual meiotic regulator. Although boule has been proposed as the ancestor for dazl/daz by gene duplication, it has been identified only in invertebrates and mammals. It has, however, remained unclear when and how the DAZ family has evolved in vertebrates.

Methodology and Principal Findings

This study was aimed at identifying and characterizing the DAZ family genes in fish as the basal vertebrate. We show that boule and dazl coexist in medaka and stickleback. Similar to the medaka dazl (Odazl), the medaka boule (Obol) is maternally supplied and segregates with primordial germ cells. Surprisingly, Obol is expressed in adult germ cells at pre-meiotic and meiotic stages of spermatogenesis and oogenesis. However, the maximal meiotic Obol expression in spermatocytes contrasts with the predominant pre-meiotic Odazl expression in spermatogonia, and the diffuse cytoplasmic Obol distribution in early oocytes contrasts with the Odazl concentration in the Balbinani''s body.

Conclusions

The identification of fish boule and dazl genes provides direct evidence for the early gene duplication during vertebrate evolution. Our finding that Obol exhibits bisexual expression in both embryonic and adult germ cells considerably extends the diversity of boule expression patterns and offers a new insight into the evolutions of DAZ family members, expression patterns and functions in animal fertility.     

DAZ Family Proteins,Key Players for Germ Cell Development

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.     

The RNA-binding specificity of the mouse Dazl protein

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 only expressed in germ cells and is necessary for meiosis. A dual approach was taken to understand the RNA-binding specificity of the Dazl protein: (i) traditional SELEX and (ii) 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 interacted specifically with Dazl in vitro. The tri-hybrid screen retrieved UTRs of known genes that may be physiological substrates of Dazl.     

Dynamic intracellular localization of Dazl protein during <Emphasis Type="Italic">Xenopus</Emphasis> germline development

Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.     

Cloning and characterization of the gene encoding the bovine BOULE protein

The Deleted in Azoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in the germ-line. The bovine Deleted in Azoospermia-like gene is a strong candidate for male cattle-yak infertility. In this work, with the goe goal to further reveal the genetic cause of male cattle-yak sterility, another bovine DAZ family gene, b-boule, was isolated and characterized. The b-boule gene is predicted to encode a polypeptide of 295 amino acids with an RNP-type RNA recognition domain. Tertiary structure analysis shows that b-boule binds specifically to polypyrimidine RNAs and might act as a nuclear ribonucleoprotein particle auxiliary factor during germ cell formation and morphological changes of germ cells. RT-PCR assays revealed that b-boule was expressed specifically in the adult testis. However, an extremely low level of expression was detected in the testis of sterile male cattle-yaks. Microstructure of the testes from sterile males showed that type A spermatogonia were the only germ cells present and that few germ cells developed further than the stage of pachytene spermatocytes. These results suggest that b-boule may function in bovine spermatogenesis, and that low levels of b-boule expression might lead to male sterility in cattle-yaks.     

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes

Background

Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method.

Results

The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs.

Conclusions

This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.

     

Post-transcriptional regulation of the meiotic Cdc25 protein Twine by the Dazl orthologue Boule

Boule, a Drosophila orthologue of the vertebrate Dazl fertility factors, is a testis-specific regulator of meiotic entry and germline differentiation. Mutations inactivating either Boule, which is an RNA-binding protein, or Twine, which is a Cdc25-type phosphatase, block meiotic entry in males. Here we show that twine and boule interact genetically. We also find that protein expression from twine messenger RNA correlates with cytoplasmic accumulation of Boule and is markedly reduced by boule mutations. Remarkably, heterologous expression of Twine rescues the boule meiotic-entry defect, indicating that the essential function of Boule at the transition from G2 to M phase during meiosis is in the control of Twine translation.     

Vasa Identifies Germ Cells and Critical Stages of Oogenesis in the Asian Seabass

Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.     

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.     

人类Boule基因在减数分裂时期的生物信息学分析

Boule（boll）基因是DAZ基因家族的一个新成员,含有一个RNA结合域和一个DAZ重复域,是人类精子发生减数分裂过程中主要调控因子。为了研究Boule基因结构及其功能,利用生物信,息学方法对Boule蛋白相关结构、相互作用蛋白及其功能进行分析和预测。结果表明,Boule蛋白存在明显的亲水区、疏水区和卷曲螺旋;不存在信号肽、跨膜结构;主要分布在细胞质、细胞核、线粒体中;二级结构以α-螺旋、延伸链、无规则卷曲所组成,并含有非正规二级结构区;作用蛋白主要为CDC25A蛋白．功能域主要包含RRM保守域。Boule蛋白在精子发生过程中第一次减数分裂的生殖细胞中特异性表达,而在减数分裂完全阻滞的睾丸组织中不表达。因此,Boule蛋白功能可能与雄性生殖细胞减数分裂相关基因表达有关。