Evolutionary comparison of the reproductive genes, DAZL and BOULE, in primates with and without DAZ

Genes of the DAZ (Deleted in AZoospermia) gene family, DAZ, DAZL (DAZ-Like), and BOULE, encode closely related RNA-binding proteins that are required for fertility in numerous organisms, yet the genomes of different organisms possess different complements of DAZ family genes. Thus, invertebrates such as flies and worms contain just a single DAZ homolog, boule, while genomes of vertebrates, other than catarrhine primates (Old World monkeys and hominids), possess both Boule and Dazl genes. Finally, catarrhine primates possess BOULE, DAZL, and DAZ genes. Since the DAZ genes arose recently in evolution in the catarrhine lineage, we sought to examine how the sequences and expression of this gene family may have changed after the introduction of a new member, DAZ. Based on previous results, we hypothesized that the introduction of a new member of the DAZ gene family into catarrhines could reduce functional constraint on DAZL. Surprisingly, however, we found that platyrrhine DAZL demonstrated significantly more sequence divergence than catarrhine DAZL (p=0.0006 for nucleotide and p=0.05 for amino acid sequence); however, comparison of K _a/K _s ratios suggests that the DAZL and BOULE genes are under similar functional constraints regardless of lineage. Thus, our data are most consistent with the hypothesis that the introduction of DAZ did not affect the evolution of DAZL or BOULE, and that a higher neutral mutation rate in platyrrhines than in catarrhines, along with the greater tolerance of DAZL for variation relative to BOULE, may be the foundation for the observed differences in sequence divergence in this gene family.     

Boule-like genes regulate male and female gametogenesis in the flatworm Macrostomum lignano

Members of the DAZ (Deleted in AZoospermia) gene family are important players in the process of gametogenesis and their dysregulation accounts for 10% of human male infertility. Boule, the ancestor of the family, is mainly involved in male meiosis in most organisms. With the exception of Drosophila and C. elegans, nothing is known on the function of boule in non-vertebrate animals. In the present study, we report on three boule orthologues in the flatworm Macrostomum lignano. We demonstrate that macbol1 and macbol2 are expressed in testes whilst macbol3 is expressed in ovaries and developing eggs. Macbol1 RNAi blocked spermatocyte differentiation whereas macbol2 showed no effect upon RNAi treatment. Macbol3 RNAi resulted in aberrant egg maturation and led to female sterility. We further demonstrated the evolutionary functional conservation of macbol1 by introducing this gene into Drosophila bol¹ mutants. Macbol1 was able to rescue the progression of fly meiotic divisions. In summary, our findings provide evidence for an involvement of boule genes in male and female gamete development in one organism. Furthermore, boule gene function is shown here for the first time in a lophotrochozoan. Our results point to a more diverse functional assignment of boule genes. Therefore, a better understanding of boule function in flatworms can help to elucidate the molecular mechanisms of and concomitant infertility in higher organisms including humans.     

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.     

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.     

Evolution of the <Emphasis Type="Italic">DAZ</Emphasis> gene and the <Emphasis Type="Italic">AZFc</Emphasis> region on primate Y chromosomes

Background  

The Azoospermia Factor c (AZFc) region of the human Y chromosome is a unique product of segmental duplication. It consists almost entirely of very long amplicons, represented by different colors, and is frequently deleted in subfertile men. Most of the AZFc amplicons have high sequence similarity with autosomal segments, indicating recent duplication and transposition to the Y chromosome. The Deleted in Azoospermia (DAZ) gene within the red-amplicon arose from an ancestral autosomal DAZ-like (DAZL) gene. It varies significantly between different men regarding to its copy number and the numbers of RNA recognition motif and DAZ repeat it encodes. We used Southern analyses to study the evolution of DAZ and AZFc amplicons on the Y chromosomes of primates.     

Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus)

The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.     

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.     

Antisense inhibition of a nuclear gene,BrDAD1, in Brassica causes male sterility that is restorable with jasmonic acid treatment

Male sterility is widely used for the production of hybrid seeds, but the use of genic male sterility is rather limited because of difficulty in maintaining homozygous male sterile plants. Recently, the DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) gene, which encodes a phospholipase A1 involved in the first step of the jasmonic acid (JA) biosynthesis pathway, was isolated from a male sterile Arabidopsis mutant. To utilize this gene in Brassica crops, we characterized the BrDAD1 gene, the putative ortholog of DAD1 in Brassica rapa. Out of 25 plants transformed with an antisense gene constructed from the BrDAD1, 3 plants showed a defect of anther dehiscence at the flower bud opening stage and produced inviable pollen. One of the three showed male sterility only, but the other two showed a delay or a lack of flower opening in addition to male sterility. The male sterile and flower-opening phenotypes were rescued by the application of JA as well as linolenic acid. Furthermore, all these characteristics were inherited to the next generation. The present results demonstrate a novel control system for hybrid seed production by the use of nuclear genes.     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distinguishing among male sterile and fertile lines of tomatoes (Lycopersicon esculentum Mill.) by means of electrophoretic protein patterns

Vertical block SDS-PAAG electrophoresis of individual tomato seeds was used to examine the potential of seed proteins as gene markers for distinguishing male sterile from fertile lines and cultivars. Seeds of three genic male sterile lines and four fertile cultivars and lines were investigated. The line 6944 is carrier of the gene ms 10 ³⁵for pollen male sterility and the lines P-1a and PPS-3 carry the gene ps-2 for positional sterility. A modified extract solution which includes 1M tris — buffer pH 7.0, 7 % SDS, dimethylformamide and distilled water was applied. Qualitative variation in the A protein locus which could be used as a marker for distinguishing male sterile from fertile tomato lines was established.     

Cloning and characterization of chicken SPATA4 gene and analysis of its specific expression

The spermatogenesis associated 4 gene (SPATA4, previously named TSARG2) was first cloned from a mouse testis cDNA library and was reported to be a candidate apoptosis-related gene in male germ cells. In this study, we cloned and characterized the SPATA4 gene from chicken (Gallus gallus). Bioinformatics analysis shows that the chicken SPATA4 gene is located on chromosome 4, is made up of six exons, and contains an 860 bp open reading frame encoding a putative protein of 250 amino acids. Further analysis of the SPATA4 gene sequence indicates that it is highly conserved between avian and mammalian species. Multi-tissue RT-PCR results indicate that the chicken SPATA4 gene is specifically expressed in the testis. Moreover, according to multi-time RT-PCR results, the expression of chicken SPATA4 occurs in a development stage-dependent pattern, and is gradually upregulated during the developmental process in chicken testis. All of these results suggest that SPATA4 may play an important role in the chicken spermatogenesis process.     

The low expression of Dmrt7 is associated with spermatogenic arrest in cattle-yak

Dmrt7 is a member of the DM domain family of genes. Dmrt7 deficiency is also a strong candidate as a cause for male cattle-yak infertility, as it is regarded as essential for male spermatogenesis, between the pachynema and diplonema stages. In our study, the coding region sequence of yak and cattle-yak Dmrt7 was cloned by molecular cloning techniques, and the sequence, conserved domains, functional sites, and secondary and tertiary structures of the Dmrt7-encoded protein were predicted and analyzed using bioinformatics methods. The coding region sequences of the Dmrt7 gene, encoding 370 amino acids, were consistent in yak and cattle-yak. The protein encoded by yak and cattle-yak Dmrt7 contains a DM domain. We detected Dmrt7 mRNA expression in testis, but not in any other tissue. Dmrt7 mRNA and protein expression was significantly higher in testis of cattle and yak than that in cattle-yak (p < 0.01). Histological analysis indicated that seminiferous tubules in male cattle-yak were highly vacuolated and contained primarily Sertoli cells and spermatogonia, while those of cattle and yak contained abundant primary spermatocytes. Male cattle-yak testis contained a significantly larger number of apoptotic cells than those in cattle and yak assessed by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) analysis. The accumulation of SCP3-positive spermatocytes indicated the arrest of spermatogenesis at the pachynema stage in the cattle-yak. These results suggest low levels of Dmrt7 expression lead to male sterility in cattle-yak. The molecular function of Dmrt7 and the regulation of its expression warrant need to be examined in future studies.     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

The second intron of AGAMOUS drives carpel- and stamen-specific expression sufficient to induce complete sterility in Arabidopsis

Gene containment technologies that prevent transgene dispersal through pollen, fruit and seed are in immediate demand to address concerns of gene flow from transgenic crops into wild species or close relatives. In this study, we isolated the enhancer element of Arabidopsis AGAMOUS that drives gene expression specifically in stamens and carpels. By fusing this AG enhancer to a minimal 35S promoter fragment, two tissue-specific promoters, fAGIP and rAGIP in forward and reverse orientations, respectively, were created and fused to the GUS reporter. Transgenic Arabidopsis plants harboring either fAGIP::GUS or rAGIP::GUS displayed similar GUS expression specifically in carpel and stamen tissues and their primordial cells. To test their utility for engineering sterility, the promoters were fused to the Diphtheria toxin A (DT-A) gene coding for a ribosome inactivating protein as well as the Barnase gene coding for an extracellular ribonuclease, and tested for tissue-specific ablation. Over 89% of AGIP::DT-A and 68% of AGIP::Barnase transgenic plants displayed specific and precise ablation of stamens and carpels and are completely sterile. These transgenic plants showed normal vegetative development with prolonged vegetative growth. To evaluate the stability of the sterile phenotype, 16 AGIP::DT-A lines underwent two consecutive cutback generations and showed no reversion of the floral phenotype. This study demonstrates a simple, precise and efficient approach to achieve absolute sterility through irreversible ablation of both male and female floral organs. This approach should have a practical application for transgene containment in ornamental, landscaping, and woody species, whose seeds and fruits are of no economic value.     

Association of the mouse infertility factor DAZL1 with actively translating polyribosomes

The DAZ (Deleted in AZoospermia) 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. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 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. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.     

Regulation by Hsp27/P53 in testis development and sperm apoptosis of male cattle (cattle-yak and yak)

Heat shock protein 27 (Hsp27)/protein 53 (P53) plays an important role in testis development and spermatozoa regulation, but the relationship between Hsp27/P53 and infertility in cattle is unclear. Here, we focus on male cattle-yak and yak to investigate the expression and localization of Hsp27/P53 in testis tissues and to explore the influence of Hsp27/P53 on infertility. In our study, a total of 54 cattle (24 cattle-yak and 30 yak) were examined. The Hsp27 and P53 messenger RNA (mRNA) of cattle-yak were cloned, and amino acid variations in Hsp27 and P53 were found; the variations led to differences in the protein spatial structure compared with yak. We used real-time quantitative polymerase chain reaction and western blot to investigate whether the expression of Hsp27/P53 mRNA and protein was different in cattle-yak and yak. We found that the expression levels of Hsp27/P53 mRNA and protein were different in the testis developmental stages and the highest expression was observed in testicles during adulthood. Moreover, the Hsp27 expression was significantly higher in yak, whereas P53 expression was higher in cattle-yak (p < 0.01). On this basis, we detected the location of Hsp27/P53 in the testis by immunohistochemistry and immunofluorescence. The results demonstrated that Hsp27 was located in spermatogenic cells at different developmental stages and mesenchymal cells of the yak testicles. However, P53 was located in the primary spermatocyte and interstitial cells of the cattle-yak testicles. In summary, our study proved that the expression of Hsp27/P53 differed across the testis developmental stages and the expression of P53 was higher in the testis of cattle-yak, which suggested that the infertility of cattle-yak may be caused by the upregulation of P53.     

Variants in Deleted in AZoospermia-Like (DAZL) are correlated with reproductive parameters in men and women

Qualitative and quantitative defects in human germ cell production that result in infertility are common and determined at least in part by genetic factors [Matzuk and Lamb, Nat Cell Biol 4(Suppl):s41–s49, 2002]. Yet, very few genes that are associated with germ cell defects in humans have been identified. In this study, we examined whether variants of the Deleted in AZoospermia-Like (DAZL) gene are associated with measures of germ cell production in three distinct populations of men and women. We identified 95 sequence variants in DAZL and further analyzed twelve single nucleotide polymorphisms (SNPs) that were present across ethnicities. We found that seven of the twelve SNPs were associated with at least one of the parameters studied (age at premature ovarian failure or menopause, total sperm count, or total motile sperm count). Surprisingly, many alleles exhibited opposing effects in men and women, which may be a result of different genetic requirements in male and female germ cells. Single SNP and haplotype analysis suggested that SNPs in the DAZL gene may act jointly to affect common reproductive characteristics in the human population. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Joyce Y. Tung and Mitchell P. Rosen have contributed equally     

Allelic relationship of four male sterility genes and nucleo-cytoplasmic interactions in the expression of male sterility in pearl millet,Pennisetum americanum (L.) leeke

Summary Male sterility genes isolated in four inbred lines of pearl millet were found allelic. The differences between male fertile and male sterile phenotypes is mainly due to a single gene. Presence of a dominant gene (Ms) resulted in male fertility and double recessiveness (ms ms) in male sterility. However, genic male sterility (GMS) in Pennisetum is not a simply inherited case of monogenic recessive condition but is influenced by cytoplasmic and several nuclear factors. In a male sterile, the stage at which the male sterility gene is expressed during the development of the male gametophyte resulting in breakdown of the cells is influenced by cytoplasmic and other nuclear factors. Two types of cytoplasm, C-1 and C-2, are recognized. Presence of any two recessive male sterility alleles in C-1 led to breakdown of male development before differentiation of an archesporium in the anther (Arc-type); in C-2 cytoplasm, degeneration started during meiosis with fusion of meiocytes and syncyte formation (Syn-type), or at post-meiotic stages terminating in abortion of microspores before first pollen mitosis (PGM type). The triggering of activity of recessive male sterility genes in C-2 cytoplasm appeared to be regulated by two nuclear factors, R ₁ and R ₂ with duplicate gene action. Recessiveness for both the R factors in C-2 cytoplasm resulted in PGM-type expression. The action of R ₁ and R ₂ is specific to C-2 cytoplasm. Mutation of cytoplasm from C-1 to C-2 and C-2 to C-1 was observed.