Heritable artificial sex chromosomes in the medaka, Oryzias latipes

Abstract

Chromosomal sex determination is widely used by vertebrates, however, only two genes have been identified as master sex-determining genes: SRY/Sry in mammals and DMY in the teleost medaka. Transfer of both genes into genetically female (XX) individuals can induce male development. However, transgenic strains have not been established in both cases because of infertility of the transgenic founders in mammals and low germline transmission rates in medaka. In this study, we used a BAC clone containing DMY in a 117 kb genomic region and two types of fluorescent marker to establish two DMY-transgenic medaka strains. In these strains, exogenous DMY is completely linked to a male phenotype and early gonadal development is not different from that of the wild-type strain. Sex-linkage analysis showed that the exogenous DMY was located on linkage group (LG) 23 in one strain and on LG 5 in the other strain, whereas the sex chromosome in medaka is on LG 1. Real-time PCR analysis indicated that these strains have multiple copies of DMY and higher DMY expression levels than the wild-type strain. These results showed that LGs 23 and 5 function as sex chromosomes in the two strains, respectively. This is not only the first example of the artificial generation of heritable sex chromosomes in vertebrates but also the first evidence showing plasticity of homomorphic sex chromosomes. This plasticity appears to be a characteristic of lower vertebrates and the underlying cause of frequent sex chromosome switching, recently reported in several fish and frog species.

Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka

“…Green fluorescent protein (GFP) was used as a fluorescent reporter under the control of the medaka olvas promoter (Tanaka et al 2001). We constructed a recombinant plasmid where vasa-GFP was inserted into the pCC1 vector in an earlier study (Otake et al 2010). In the present study, we amplified this region using PCR and the following primer sets: 5′-TAG CGA TGA GCT CGG ACT TCC ATT GTT CA-3′ and 5′-CGT AAT CAT GGT CAT AGC TGT TTC CTG TGT-3′.…”

Heritable artificial sex chromosomes in medaka: a leap towards real-time observation of sex chromosome evolution

“…Sex-linkage analysis of the transgenic strains of Otake et al indicated that exogenous DMY was located on LG5 in one strain, and LG23 in another (Otake et al, 2010). Real-time PCR experiments confirmed that DMY is only expressed in the testes of transgenic strains, and that they have multiple copies of DMY and higher DMY expression levels than wild-type medaka.…”

“…Even the neo-Y chromosome of Drosophila miranda is 41 million years old (Bachtrog and Charlesworth, 2002), whereas 'young' sex chromosomes of fish are in excess of 10 million years old (for example, Tanaka et al, 2007), so we can only bear indirect witness to their evolution. As reported in this issue, researchers in Japan have developed a more practicable alternative to time travel by creating the first vertebrate heritable artificial sex chromosomes in the medaka, Oryzias latipes (Otake et al, 2010). In doing so, they open a new and exciting chapter in sex chromosome research.…”

“…Previous attempts to generate DMY transgenic medaka showed testis differentiation with spermatogenesis, but low transmission rates, hence transgenic strains could not be established (Matsuda et al, 2007). Otake et al (2010) built on the work of Matsuda et al (2007) by integrating fluorescent markers into the transgenic region. This allowed them to screen transgene-positive XX male fish and identify fry that had inherited, and were therefore good candidates for passing on the novel sex chromosome to their own offspring.…”

An autosomal locus controls sex reversal in interspecific XY hybrids of the medaka fishes

“…Dmy is the Y-specific paralog of the autosomal Dmrt1 gene which appears to be involved in male sexual development in vertebrates (Nanda et al, 2002). Mutations with impaired Dmy function cause XY sex reversal (Shinomiya et al, 2004;Otake et al, 2006Otake et al, , 2008 and the presence of an exogenous Dmy gene induces XX sex reversal (Matsuda et al, 2007;Otake et al, 2009), suggesting that this gene is necessary and sufficient to induce testicular development. Dmy contains a DNA-binding motif (the DM domain), and is thus thought to encode a putative transcription factor that activates or represses downstream target genes to regulate sexual differentiation of the bipotential gonad.…”