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

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.

Untitled

“…The expression level in our study differed from tissues, little or no expression was found in most of the digestive organs except for spleen; on contrary, high expression was found in other parts such as brain, gills and spleen. This result is similar to a previous research on trout that constitutive expression of TGF-β1 was found in brain, gills, spleen, head kidney and peripheral blood leucocytes except in liver (Li et al, 2015).…”

“…Specific expression of TGF-β1 relative to β-actin was analysed using densitometry. super family may control sex associated with growth (Li et al, 2015). Brain, muscle and other organs have been identified greatly contributing to fish growth.…”

“…Thus, a number of studies on the expression of immune-regulatory genes in fish infected with various bacterial, viral as well as parasitic pathogens, and their involvement in specific disease processes have been performed (Border & Noble, 1994;Li et al, 2015;Wu & Hill, 2009). Most of the immune-related cells are regulated by transforming growth factor-β (TGF-β) directly or indirectly, such as in the emergence of diseases, participation stress signal transduction and cell response (Gorelik & Flavell, 2000;Li et al, 2015).…”

Demonstration of viability and fertility and development of a molecular tool to identify YY supermales in a fish with both genotypic and environmental sex determination

“…In addition to being very fast, the qPCR showed 100% agreement with the results of genotypic sex inferred by progeny test, which demonstrates its reliability and potential usefulness for field studies. An alternative approach is the amplification of Y and X chromosome‐specific markers by nonquantitative PCR as has been used for distinction of YY supermales in yellow catfish Pelteobagrus fulvidraco (Wang, Mao, Chen, Liu, & Gui, 2009) and Nile tilapia O. niloticus (Li et al., 2015). However, in these cases, specific markers for both sex chromosomes are required, limiting the application of this method to a handful of species for which such markers have been developed.…”

The Reversible Sex of Gonochoristic Fish: Insights and Consequences

“…Despite having a strong TSD, another pejerrey, O. bonariensis, also expressed amhy at early stages of male promoting temperatures followed by the expression of the autosomal amha gene, showing the coexistence of both temperature and genetic sex determination in this species [Yamamoto et al, 2014]. Two tandem Y copies of the amh gene have been identified in the Nile tilapia with a 233-bp deleted region in exon VII and a 5-bp insertion in exon VI that leads to a truncated amh gene lacking the TGF-β domain [Eshel et al, 2014;Li et al, 2015], defined as amhΔY, and another amhy having a missense SNP which may be determining sex in some strains, notably the Japanese strain [Li et al, 2015]. In several pufferfish (Takifugu) species an allelic variation in the amhr2 gene, the amh receptor, has been shown to be responsible for maleness [Kamiya et al, 2012].…”