The forkhead factor Foxl2 participates in the ovarian differentiation of Chinese soft-shelled turtle Pelodiscus sinensis.

The forkhead transcription factor Foxl2 plays a major role in ovarian development and function in mice and fish, and acts as a female sex-determining gene in goat. Its functional role in the sex determination and gonadal differentiation has not yet been investigated in reptiles. Here, we characterized Foxl2 gene in Chinese soft-shelled turtle Pelodiscus sinensis, exhibiting ZZ/ZW sex chromosomes. Foxl2 exhibited a female-specific embryonic expression pattern throughout the critical sex determination periods in P. sinensis. The expression of Foxl2 was induced at early stage in ZZ embryonic gonads that were feminized by estrogen treatment. Most importantly, Foxl2 knockdown in ZW embryos by RNA interference resulted in female-to-male sex reversal, characterized by obvious masculinization of gonads, significant up-regulation of testicular markers Dmrt1 and Sox9, and remarkable down-regulation of ovarian regulator Cyp19a1. Conversely, gain-of-function study showed that overexpression of Foxl2 in ZZ embryos led to largely feminized genetic males, production of Cyp19a1, and a decline in Dmrt1 and Sox9. These findings demonstrate that Foxl2 is both necessary and sufficient to initiate ovarian differentiation in P. sinensis, thereby acting as a key upstream regulator of the female pathway in a reptilian species.

The Role of Anti-Müllerian Hormone in Testis Differentiation Reveals the Significance of the TGF-ß Pathway in Reptilian Sex Determination.

Anti-Müllerian hormone (Amh, or Müllerian-inhibiting substance, Mis), a member of TGF-ß superfamily, has been well documented in some vertebrates as initiator or key regulator in sexual development, and particularly in fish. However, its functional role has not yet been identified in reptiles. Here, we characterized the Amh gene in the Chinese soft-shelled turtle Pelodiscus sinensis, a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The messenger RNA of Amh was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited a male-specific expression pattern throughout embryogenesis. Moreover, Amh was rapidly upregulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, Amh loss of function by RNA interference led to complete feminization of genetic male (ZZ) gonads, suppression of the testicular marker Sox9, and upregulation of the ovarian regulator Cyp19a1 Conversely, overexpression of Amh in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of a testis structure, ectopic activation of Sox9, and a remarkable decline in Cyp19a1 Collectively, these findings provide the first solid evidence that Amh is both necessary and sufficient to drive testicular development in a reptilian species, P. sinensis, highlighting the significance of the TGF-ß pathway in reptilian sex determination.

Author Correction: Dmrt1 is required for primary male sexual differentiation in Chinese soft-shelled turtle Pelodiscus sinensis.

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Dmrt1 is required for primary male sexual differentiation in Chinese soft-shelled turtle Pelodiscus sinensis.

In vertebrates, the primary sex-determining signals that initiate sexual development are remarkably diverse, ranging from complete genetic to environmental cues. However, no sex determination-related genes have been functionally identified in reptiles. Here, we characterized a conserved DM domain gene, Dmrt1, in Chinese soft-shelled turtle Pelodiscus sinensis (P. sinensis), which exhibits ZZ/ZW sex chromosomes. Dmrt1 exhibited early male-specific embryonic expression, preceding the onset of gonadal sex differentiation. The expression of Dmrt1 was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment. Dmrt1 knockdown in ZZ embryos by RNA interference resulted in male to female sex reversal, characterized by obvious feminization of gonads, significant down-regulation of testicular markers Amh and Sox9, and remarkable up-regulation of ovarian regulators, Cyp19a1 and Foxl2. Conversely, ectopic expression of Dmrt1 led to largely masculinized genetic females, production of Amh and Sox9, and a decline in Cyp19a1 and Foxl2. These findings demonstrate that Dmrt1 is both necessary and sufficient to initiate testicular development, thereby acting as an upstream regulator of the male pathway in P. sinensis.

Heterologous expression and purification of aldehyde dehydrogenase gene from Bacillus halodurans XJU-1.

BACKGROUND: For its function of catalysing the reaction of aldehyde to acetic acid, ALDH could be applied to antialcoholismic drug development. However, both of the production and activity of natural ALDH are too low to meet the demand. OBJECTIVE: To empirically explore whether aldh gene in Bacillus halodurans XJU-1 could be highly expressed in E.coli BL21(DE3) and to investigate the purification of recombinant protein ALDH. METHODS: The aldh gene in Bacillus halodurans XJU-1 was cloned into prokaryotic expression vector pET30b(+), then transformed into E.coli BL21(DE3) to induce expression of ALDH. Immobilized metal ion affinity chromatography (IMAC) was applied in the separation and purification for ALDH proteins. RESULTS: The recombinant vector for aldh gene was constructed and expressed in BL21(DE3) successfully. The ALDH recombinant protein was purified by ammonium sulfate precipitation and IMAC, and obtained 30.1% of the recovery with a purification factor 8.81. CONCLUSIONS: The value of these findings, as well as wider implications for increasing the yield and the activity of ALDH, is meaningful.