Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry , leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis , in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9 . The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Using a comprehensive transcriptome analysis, a Z chromosome-linked chicken homolog of hemogen ( cHEMGN ) was identified and shown to be specifically involved in testis differentiation in early chicken embryos. Hemogen [ Hemgn in mice, EDAG (erythroid differentiation-associated gene protein) in humans] was recently characterized as a hematopoietic tissue-specific gene encoding a transcription factor that regulates the proliferation and differentiation of hematopoietic cells in mammals. In chicken, cHEMGN was expressed not only in hematopoietic tissues but also in the early embryonic gonad of male chickens. The male-specific expression was identified in the nucleus of (pre)Sertoli cells after the sex determination period and before the expression of SOX9 (SRY-box 9). The expression of cHEMGN was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment. ZW embryos overexpressing cHEMGN , generated by infection with retrovirus carrying cHEMGN , showed masculinized gonads. These findings suggest that cHEMGN is a transcription factor specifically involved in chicken sex determination.

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.

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.