A Tandem Duplicate of Anti-Müllerian Hormone with a Missense SNP on the Y Chromosome Is Essential for Male Sex Determination in Nile Tilapia, Oreochromis niloticus

PLoS Genet. 2015 Nov 20;11(11):e1005678. doi: 10.1371/journal.pgen.1005678. eCollection 2015 Nov.

Abstract

Variation in the TGF-β signaling pathway is emerging as an important mechanism by which gonadal sex determination is controlled in teleosts. Here we show that amhy, a Y-specific duplicate of the anti-Müllerian hormone (amh) gene, induces male sex determination in Nile tilapia. amhy is a tandem duplicate located immediately downstream of amhΔ-y on the Y chromosome. The coding sequence of amhy was identical to the X-linked amh (amh) except a missense SNP (C/T) which changes an amino acid (Ser/Leu92) in the N-terminal region. amhy lacks 5608 bp of promoter sequence that is found in the X-linked amh homolog. The amhΔ-y contains several insertions and deletions in the promoter region, and even a 5 bp insertion in exonVI that results in a premature stop codon and thus a truncated protein product lacking the TGF-β binding domain. Both amhy and amhΔ-y expression is restricted to XY gonads from 5 days after hatching (dah) onwards. CRISPR/Cas9 knockout of amhy in XY fish resulted in male to female sex reversal, while mutation of amhΔ-y alone could not. In contrast, overexpression of Amhy in XX fish, using a fosmid transgene that carries the amhy/amhΔ-y haplotype or a vector containing amhy ORF under the control of CMV promoter, resulted in female to male sex reversal, while overexpression of AmhΔ-y alone in XX fish could not. Knockout of the anti-Müllerian hormone receptor type II (amhrII) in XY fish also resulted in 100% complete male to female sex reversal. Taken together, these results strongly suggest that the duplicated amhy with a missense SNP is the candidate sex determining gene and amhy/amhrII signal is essential for male sex determination in Nile tilapia. These findings highlight the conserved roles of TGF-β signaling pathway in fish sex determination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Anti-Mullerian Hormone / genetics*
  • Female
  • Gene Duplication*
  • Genetic Linkage
  • Male
  • Mutation, Missense*
  • Polymorphism, Single Nucleotide*
  • Sex Determination Processes*
  • Tilapia / genetics*
  • Y Chromosome*

Substances

  • Anti-Mullerian Hormone

Grant support

This work was supported by grant 2011AA100404 from the National High Technology Research and Development Program (863 program) of China; grants 91331119 from the National Natural Science Foundation of China; grant 20130182130003 from the Specialized Research Fund for the Doctoral Program of Higher Education of China; grant cstc2013kjrc-tdjs80003 from the Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission; grant 2012CB723205 from the National Basic Research Program of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.