Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis.

Homomorphic sex chromosomes and rapid turnover of sex-determining genes can complicate establishing the sex chromosome system operating in a given species. This difficulty exists in Xenopus tropicalis, an anuran quickly becoming a relevant model for genetic, genomic, biochemical, and ecotoxicological research. Despite the recent interest attracted by this species, little is known about its sex chromosome system. Direct evidence that females are the heterogametic sex, as in the related species Xenopus laevis, has yet to be presented. Furthermore, X. laevis' sex-determining gene, DM-W, does not exist in X. tropicalis, and the sex chromosomes in the two species are not homologous. Here we identify X. tropicalis' sex chromosome system by integrating data from (i) breeding sex-reversed individuals, (ii) gynogenesis, (iii) triploids, and (iv) crosses among several strains. Our results indicate that at least three different types of sex chromosomes exist: Y, W, and Z, observed in YZ, YW, and ZZ males and in ZW and WW females. Because some combinations of parental sex chromosomes produce unisex offspring and other distorted sex ratios, understanding the sex-determination systems in X. tropicalis is critical for developing this flexible animal model for genetics and ecotoxicology.

Cytogenetic Analysis in the Toad Species Bufo spinosus, Bufotes viridis and Epidalea calamita (Anura, Bufonidae) from the Mediterranean Area.

Taxonomy in Bufonidae witnessed notable transformations. Bufotes viridis and Epidalea calamita, previously included in genus Bufo, were relocated in other genera, while the genus Bufo was restricted to members of the earlier Bufo bufo group. On the other hand, Bufo bufo sensu lato now includes four species: Bufo bufo, Bufo spinosus, Bufo verrucosissimus and Bufo eichwaldi. In this study, we examined three species of three Bufonidae genera (B. spinosus, B. viridis and E. calamita) by conventional (C-banding and Ag-NOR staining) and molecular (in situ hybridization with probes for telomeric repeats and rDNA loci, and genomic in situ hybridization (GISH)) cytogenetic methods. C-banding patterns are reported for the first time for B. spinosus and E. calamita populations from Iberian Peninsula and for B. viridis from Greece, and reveal several differences with the reported C-banded karyotypes described for other European populations of these species. Silver staining shows size heteromorphisms of the signals at the Nucleolar Organizing Region (NOR). By contrast, FISH with ribosomal probes only reveal size heteromorphism of rDNA sequences in E. calamita, suggesting that the differences observed after silver staining in B. spinosus and B. viridis should be attributed to differences in chromosomal condensation and/or gene activity rather than to differences in the copy number for ribosomal genes. Regarding telomeric repeats, E. calamita is the only species with interstitial telomeric sequences (ITS) located on centromeric regions, probably originated by accumulation of telomeric sequences in the centromeric heterochromatin. Finally, we analyzed the composition and distribution of repetitive sequences by genome in situ hybridization. These experiments reveal the accumulation of repetitive sequences in centromeric regions of the three species, although these sequences are not conserved when species from different genera are compared.

Identification and characterization of a new family of long satellite DNA, specific of true toads (Anura, Amphibia, Bufonidae).

Amphibians have some of the most variable genome sizes among vertebrates. Genome size variation has been attributed to repetitive and noncoding DNA, including satellite repeats, transposable elements, introns, and nuclear insertions of viral and organelle DNA. In vertebrates, satellite DNAs have been widely described in mammals, but few molecular studies have been carried out in amphibians. Here, we provide a detailed characterization of a new family of satellite DNA, present in all 15 examined species of the family Bufonidae. Southern-blot analysis and PCR reveal that this satellite is formed by monomers of 807 bp, is organized in tandem arrays, and has an AT-content of 57.4%. Phylogenetic analyses show that most clades exhibit species-specific variances, indicating that this satellite DNA has evolved by concerted evolution. The homogenization/fixation process is heterogeneous in Bufonidae, where the genera Bufo and Bufotes do not show species-specific differences, while populations from Rhinella marina exhibit population-specific changes. Additionally, variants of this satellite DNA have been identified in Duttaphrynus melanostictus and R. marina, supporting the 'library hypothesis' (a set, 'library', of satellite DNAs is shared by a species group). Physical mapping in Bufo bufo, Bufo spinosus, Epidalea calamita and Bufotes viridis provides evidence that this repetitive DNA is not dispersed in the karyotype, but accumulated in pericentromeric regions of some chromosomal pairs. This location, together with its presence in the transcriptomes of bufonids, could indicate a role in centromere function or heterochromatin formation and maintenance.

Testis Development and Differentiation in Amphibians.

Sex is determined genetically in amphibians; however, little is known about the sex chromosomes, testis-determining genes, and the genes involved in testis differentiation in this class. Certain inherent characteristics of the species of this group, like the homomorphic sex chromosomes, the high diversity of the sex-determining mechanisms, or the existence of polyploids, may hinder the design of experiments when studying how the gonads can differentiate. Even so, other features, like their external development or the possibility of inducing sex reversal by external treatments, can be helpful. This review summarizes the current knowledge on amphibian sex determination, gonadal development, and testis differentiation. The analysis of this information, compared with the information available for other vertebrate groups, allows us to identify the evolutionarily conserved and divergent pathways involved in testis differentiation. Overall, the data confirm the previous observations in other vertebrates-the morphology of the adult testis is similar across different groups; however, the male-determining signal and the genetic networks involved in testis differentiation are not evolutionarily conserved.

Sex Differentiation in Amphibians: Effect of Temperature and Its Influence on Sex Reversal.

The role of environmental factors in sexual differentiation in amphibians is not new. The effect of hormones or hormone-like compounds is widely demonstrated. However, the effect of temperature has traditionally been regarded as something anecdotal that occurs in extreme situations and not as a factor to be considered. The data currently available reveal a different situation. Sexual differentiation in some amphibian species can be altered even by small changes in temperature. On the other hand, although not proven, it is possible that temperature is related to the appearance of sex-reversed individuals in natural populations under conditions unrelated to environmental contaminants. According to this, temperature, through sex reversal (phenotypic sex opposed to genetic sex), could play an important role in the turnover of sex-determining genes and in the maintenance of homomorphic sex chromosomes in this group. Accordingly, and given the expected increase in global temperatures, growth and sexual differentiation in amphibians could easily be affected, altering the sex ratio in natural populations and posing major conservation challenges for a group in worldwide decline. It is therefore particularly urgent to understand the mechanism by which temperature affects sexual differentiation in amphibians.

Interaction between sex-determining genes from two species: clues from Xenopus hybrids.

Hybrids provide an interesting model to study the evolution of sex-determining genes and sex chromosome systems as they offer the opportunity to see how independently evolving sex-determining pathways interact in vivo. In this context, the genus Xenopus represents a stimulating model, since species with non-homologous sex chromosomes and different sex-determining genes have been identified. In addition, the possibility of interspecies breeding is favoured in this group, which arose by alloploidization events, with species ploidy ranging from 2n = 2x = 20 in X. tropicalis (the only diploid representative of the genus) to 2n = 12x = 108 in X. ruwenzoriensis. To study how two sex-determining genes interact in vivo, X. laevis × X. tropicalis hybrids were produced. Gonadal differentiation in these hybrids revealed that the dm-w gene is dominant over X. tropicalis male-determining sex chromosomes (Y or Z), even though the Y chromosome is dominant in X. tropicalis (Y > W>Z). In the absence of the dm-w gene (the Z chromosome from X. laevis is present), the W chromosome from X. tropicalis is able to trigger ovarian development. Testicular differentiation will take place in the absence of W chromosomes from any of the parental species. The dominance/recessivity relationships between these sex-determining loci in the context of either parental genome remains unknown. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae).

Xenopus laevis and its diploid relative, Xenopus tropicalis, are the most used amphibian models. Their genomes have been sequenced, and they are emerging as model organisms for research into disease mechanisms. Despite the growing knowledge on their genomes based on data obtained from massive genome sequencing, basic research on repetitive sequences in these species is lacking. This study conducted a comparative analysis of repetitive sequences in X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) with Cot DNA of both species revealed a conserved enrichment of repetitive sequences at the ends of the chromosomes in these Xenopus species. The repeated sequences located on the short arm of chromosome 3 from X. tropicalis were not related to the sequences on the short arm of chromosomes 3L and 3S from X. laevis, although these chromosomes were homoeologous, indicating that these regions evolved independently in these species. Furthermore, all the other repetitive sequences in X. tropicalis and X. laevis may be species-specific, as they were not revealed in cross-species hybridizations. Painting experiments in X. laevis with chromosome 7 from X. tropicalis revealed shared sequences with the short arm of chromosome 3L. These regions could be related by the presence of the nucleolus organizer region (NOR) in both chromosomes, although the region revealed by chromosome painting in the short arm of chromosome 3L in X. laevis did not correspond to 18S + 28S rDNA sequences, as they did not colocalize. The identification of these repeated sequences is of interest as they provide an explanation to some problems already described in the genome assemblies of these species. Furthermore, the distribution of repetitive DNA in the genomes of X. laevis and X. tropicalis might be a valuable marker to assist us in understanding the genome evolution in a group characterized by numerous polyploidization events coupled with hybridizations.

Expanding the classical paradigm: what we have learnt from vertebrates about sex chromosome evolution.

Until recently, the field of sex chromosome evolution has been dominated by the canonical unidirectional scenario, first developed by Muller in 1918. This model postulates that sex chromosomes emerge from autosomes by acquiring a sex-determining locus. Recombination reduction then expands outwards from this locus, to maintain its linkage with sexually antagonistic/advantageous alleles, resulting in Y or W degeneration and potentially culminating in their disappearance. Based mostly on empirical vertebrate research, we challenge and expand each conceptual step of this canonical model and present observations by numerous experts in two parts of a theme issue of Phil. Trans. R. Soc. B. We suggest that greater theoretical and empirical insights into the events at the origins of sex-determining genes (rewiring of the gonadal differentiation networks), and a better understanding of the evolutionary forces responsible for recombination suppression are required. Among others, crucial questions are: Why do sex chromosome differentiation rates and the evolution of gene dose regulatory mechanisms between male versus female heterogametic systems not follow earlier theory? Why do several lineages not have sex chromosomes? And: What are the consequences of the presence of (differentiated) sex chromosomes for individual fitness, evolvability, hybridization and diversification? We conclude that the classical scenario appears too reductionistic. Instead of being unidirectional, we show that sex chromosome evolution is more complex than previously anticipated and principally forms networks, interconnected to potentially endless outcomes with restarts, deletions and additions of new genomic material. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Origin and spread of the SRY gene on the X and Y chromosomes of the rodent Microtus cabrerae: role of L1 elements.

In the rodent species Microtus cabrerae, males as well as females present several copies of the SRY gene, a single-copy gene located on the Y chromosome in most mammals. Using different PCR approaches, we have characterized the sequence, structure, and organization of the SRY copies and their flanking regions distributed on the X and Y chromosomes of this species. All copies of SRY analyzed, including those from the Y chromosome, proved to be nonfunctional pseudogenes, as they have internal stop codons. In addition, we demonstrated the association of SRY pseudogenes with different fragments of L1 and LTR retroelements in both sex chromosomes of M. cabrerae. Examining the possible origin of SRY pseudogene and retroposons association, we propose that retroposons could have been involved in the mechanism of SRY gene amplification on the Y chromosome and in the transference of the Y-linked SRY copies to the X-chromosome heterochromatin.

Expression-based strategies for discovery of genes involved in testis and ovary development.

In recent years, strategies for gene identification based on differential gene expression have become increasingly popular, due in part to the development of microarray technology. These strategies are particularly well suited to the identification of genes involved in sex determination and gonadal development, which unlike the development of other organ systems, proceeds along two very different alternative courses, depending on the sex of the embryo. We have used a high-throughput, array-based expression screen to identify several genes expressed sex-specifically in developing mouse gonads. One of these, vanin 1, appears to play a role in mediating migration of mesonephric cells into the male genital ridge. Progress in characterizing other genes arising from the screen is discussed.

The karyotype and 5S rRNA genes from Spanish individuals of the bat species Rhinolophus hipposideros (Rhinolophidae; Chiroptera).

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.

Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y(DOM) sex reversal.

The phenomenon of B6-Y(DOM) sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (B6) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y(DOM) sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y(DOM) sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary.

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries.

Extensive vascularization of developing mouse ovaries revealed by caveolin-1 expression.

Expression screening for genes preferentially expressed in mouse fetal ovaries relative to testes identified Cav-1 as a candidate female-specific gene. Cav-1 encodes caveolin-1, a component of the cell membrane invaginations known as caveolae, which are involved in lipid regulation and signal transduction. In situ hybridization revealed high levels of Cav-1 mRNA in developing ovaries, compared with moderate or low levels in testes. Analysis of caveolin-1 protein distribution by immunofluorescence showed this difference to be due to the development of a dense and complex vascular network in the developing ovary. These observations point to a higher degree of differentiation and organization of the early stage mammalian ovary than previously suspected.

Sex chromosomes pairing in two Arvicolidae species: Microtus nivalis and Arvicola sapidus.

Arvicolid rodents present both synaptic and asynaptic sex chromosomes. We analyzed the pairing behaviour of sex chromosomes in two species belonging to this rodent group (Microtus nivalis and Arvicola sapidus). At pachynema, the sex chromosomes of both species paired in a small region while the rest remain unsynapsed. Consequently at metaphase I, sex chromosomes present end-to-end association. Thus, the pairing behaviour of sex chromosomes in these species is very similar to that previously described for other arvicolid rodents and for most mammals. According to this, we propose that synaptic sex chromosomes were the ancestral condition in the family Arvicolidae, including the genus Microtus. The phylogenetic origin of the asynaptic sex chromosomes in the genus Microtus would have arisen once in the lineage that originated the species M. arvalis/agrestis and related species, while the lineage that originated the species M. oeconomous and related species conserved synaptic chromosomes. Furthermore, the phylogenetic relationships between the genus Microtus, Chionomys and Pitymys are discussed in relation to the synaptic behaviour of sex chromosomes.

Mapping the SRY gene in Microtus cabrerae: a vole species with multiple SRY copies in males and females.

The SRY gene is a single-copy, male-specific gene, located on the Y chromosome in most mammals. However, recently we have described the presence of multiple polymorphic copies of this gene in both males and females of the vole species Microtus cabrerae. Here, we present the chromosomal localization of SRY gene copies in this species by fluorescent in situ hybridization (FISH). This technique localized these gene copies in the short arm, and hence in the euchromatic region, of the Y chromosome. Furthermore, several copies of the SRY gene are located on the X chromosome. These copies are spread along the entire heterochromatic region of the X chromosome, occupying the whole short arm, the centromeric region, and the pericentromeric region of the long arm.