Ancestral Y-linked genes were maintained by translocation to the X and Y chromosomes fused to an autosomal pair in the Okinawa spiny rat Tokudaia muenninki

Two species of the genus Tokudaia lack the Y chromosome and SRY, but several Y-linked genes have been rescued by translocation or transposition to other chromosomes. Tokudaia muenninki is the only species in the genus that maintains the Y owing to sex chromosome-autosome fusions. According to previous studies, many SRY pseudocopies and other Y-linked genes have evolved by excess duplication in this species. Using RNA-seq and RT-PCR, we found that ZFY, EIF2S3Y, TSPY, UTY, DDX3Y, USP9Y, and RBMY, but not UBA1Y, had high deduced amino acid sequence similarity and similar expression patterns with other rodents, suggesting that these genes were functional. Based on FISH and quantitative real-time PCR, all of the genes except for UTY and DDX3Y were amplified on the X and Y chromosomes with approximately 10-66 copies in the male genome. In a comparative analysis of the 372.4-kb BAC sequence and Y-linked gene transcripts from T. muenninki with the mouse Y genomic sequence, we observed that multiple-copy genes in the ancestral Y genome were nonfunctional, indicating that the gene functions were assumed by amplified copies. We also found a LTR sequence at the distal end of a SRY duplication unit, suggesting that unequal sister chromatid exchange mediated by retrotransposable elements could have been involved in SRY amplification. Our results revealed that the Y-linked genes were rescued from degeneration via translocations to other sex chromosomal regions and amplification events in T. muenninki

Initiation of recombination suppression and PAR formation during the early stages of neo-sex chromosome differentiation in the Okinawa spiny rat, Tokudaia muenninki

Background: Sex chromosomes of extant eutherian species are too ancient to reveal the process that initiated sex-chromosome differentiation. By contrast, the neo-sex chromosomes generated by sex-autosome fusions of recent origin in Tokudaia muenninki are expected to be evolutionarily ‘young’, and therefore provide a good model in which to elucidate the early phases of eutherian sex chromosome evolution. Here we describe the genomic evolution of T. muenninki in neo-sex chromosome differentiation. Results: FISH mapping of a T. muenninki male, using 50 BAC clones as probes, revealed no chromosomal rearrangements between the neo-sex chromosomes. Substitution-direction analysis disclosed that sequence evolution toward GC-richness, which positively correlates with recombination activity, occurred in the peritelomeric regions, but not middle regions of the neo-sex chromosomes. In contrast, the sequence evolution toward AT-richness was observed in those pericentromeric regions. Furthermore, we showed genetic differentiation between the pericentromeric regions as well as an accelerated rate of evolution in the neo-Y region through the detection of male-specific substitutions by gene sequencing in multiple males and females, and each neo-sex–derived BAC sequencing. Conclusions: Our results suggest that recombination has been suppressed in the pericentromeric region of neo-sex chromosomes without chromosome rearrangement, whereas high levels of recombination activity is limited in the peritelomeric region of almost undifferentiated neo-sex chromosomes. We conclude that PAR might have been formed on the peritelomeric region of sex chromosomes as an independent event from spread of recombination suppression during the early stages of sex chromosome differentiation

Paired Activating and Inhibitory Immunoglobulin-like Receptors, MAIR-I and MAIR-II, Regulate Mast Cell and Macrophage Activation

Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses

B Chromosomes Have a Functional Effect on Female Sex Determination in Lake Victoria Cichlid Fishes

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes

Molecular mechanism of male differentiation is conserved in the SRY-absent mammal, Tokudaia osimensis

The sex-determining gene SRY induces SOX9 expression in the testes of eutherian mammals via two pathways. SRY binds to testis-specific enhancer of Sox9 (TESCO) with SF1 to activate SOX9 transcription. SRY also up-regulates ER71 expression, and ER71 activates Sox9 transcription. After the initiation of testis differentiation, SOX9 enhances Amh expression by binding to its promoter with SF1. SOX8, SOX9 and SOX10, members of the SOXE gene family, also enhance the activities of the Amh promoter and TESCO. In this study, we investigated the regulation of these sexual differentiation genes in Tokudaia osimensis, which lacks a Y chromosome and the SRY gene. The activity of the AMH promoter was stimulated by SOXE genes and SF1. Mutant AMH promoters, with mutations in its SOX and SF1 binding sites, did not show significant activity by SOX9 and SF1. These results indicate that AMH expression was regulated by the binding of SOX9 and SF1. By contrast, SOXE genes could not enhance TESCO activity. These results indicate that TESCO enhancer activity was lost in this species. Furthermore, the activity of the SOX9 promoter was enhanced by ER71, indicating that ER71 may play an important role in the testis-specific expression of SOX9

Knockdown of DEAD-box helicase 4 (DDX4) decreases the number of germ cells in male and female chicken embryonic gonads

DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted invitro and invivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways

Research Note: Diethylstilbestrol reduces primordial germ cells in male Japanese quail

ABSTRACT: This study investigated the detrimental effects of diethylstilbestrol (DES), an estrogenic endocrine-disrupting chemical, on the viability of primordial germ cells (PGCs), embryonic precursors of germ cells, in Japanese quail. We injected 50 or 100 nmol DES solubilized in sesame oil into the yolk of stage X embryos and assessed changes in the population and cell cycle properties of circulating PGCs in blood vessels and gonadal PGCs after 2.5- and 7-day incubations, respectively. Liquid chromatography tandem mass spectrometer and Western blotting analyses identified DEAD-box polypeptide 4 (DDX4) and proliferating cell nuclear antigen (PCNA) as a stem cell marker and proliferation marker of quail PGCs, respectively. Immunochemical analyses revealed significant decreases in the number of DDX4- and PCNA-positive blood-circulating PGCs in males treated with 50 and 100 nmol DES than in the oil-treated control group. These reductions were not observed in females. Furthermore, the number of DDX4-positive gonadal PGCs was smaller in males treated with 50 and 100 nmol DES than in the control group, and these reductions were not observed in females. The protein expression of the Sertoli cell marker showed normal testis development in DES-treated embryos on d 7. These results demonstrate the potentially cytotoxic effects of DES on male germ cells, namely, the inhibition of cell cycle progression and induction of apoptosis in Japanese quail

Mutations in the Testis-Specific Enhancer of SOX9 in the SRY Independent Sex-Determining Mechanism in the Genus Tokudaia

SRY (sex-determining region Y) is widely conserved in eutherian mammals as a sex-determining gene located on the Y chromosome. SRY proteins bind to the testis-specific enhancer of SOX9 (TES) with SF1 to upregulate SOX9 expression in undifferentiated gonads of XY embryos of humans and mice. The core region within TES, named TESCO, is an important enhancer for mammalian sex determination. We show that TESCO of the genus Tokudaia lost enhancer activity caused by mutations in its SRY and SF1 binding sites. Two species of Tokudaia do not have the Y chromosome or SRY, and one species has multiple SRYs located on the neo-Y chromosome consisting of the Y fused with an autosome. The sequence of Tokudaia TESCO exhibited more than 83% identity with mouse TESCO, however, nucleotide substitution(s) were found in two out of three SRY binding sites and in five out of six SF1 binding sites. TESCO of all species showed low enhancer activity in cells co-transfected with SRY and SF1, and SOX9 and SF1 in reporter gene assays. Mutated TESCO, in which nucleotide substitutions found in SRY and SF1 binding sites were replaced with mouse sequence, recovered the activity. Furthermore, SRYs of the SRY-positive species could not activate the mutated TESCO or mouse TESCO, suggesting that SRYs lost function as a sex-determining gene any more. Our results indicate that the SRY dependent sex-determining mechanism was lost in a common ancestor of the genus Tokudaia caused by nucleotide substitutions in SRY and SF1 binding sites after emergence of a new sex-determining gene. We present the first evidence for an intermediate stage of the switchover from SRY to a new sex-determining gene in the evolution of mammalian sex-determining mechanism

Identification of chromosome rearrangements between the laboratory mouse (Mus musculus) and the Indian spiny mouse (Mus platythrix) by comparative FISH analysis

Comparative chromosome painting was applied to the Indian spiny mouse (Mus platythrix) with mouse (M. musculus) chromosome-specific probes for understanding the process of chromosome rearrangements between the two species. The chromosome locations of the 5S and 18S-28S ribosomal RNA genes and the order of the 119 and Tcp-1 genes in the In(17)2 region of the t-complex were also compared. All the painting probes were successfully hybridized to the Indian spiny mouse chromosomes, and a total of 27 segments homologous to mouse chromosomes were identified. The comparative FISH analysis revealed that tandem fusions were major events in the chromosome evolution of the Indian spiny mouse. In addition, other types of chromosome rearrangements, i.e. reciprocal translocations and insertions, were also included