Identification of male-specific amh duplication, sexually differentially expressed genes and microRNAs at early embryonic development of Nile tilapia (Oreochromis niloticus)

Abstract

BackgroundThe probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development.ResultsArtificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY ‘supermales’ resulted in all-female and all-male embryos, respectively. RNA of pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20β-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10−9). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20β-hsd and amh. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exonVII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-β domain). amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns by a false discovery rate of p < 0.05. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20β-hsd, down-regulated in males.ConclusionsThis study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-β domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-774) contains supplementary material, which is available to authorized users.

Simultaneous exposure to estrogen and androgen resulted in feminization and endocrine disruption

“…In fact, high doses of E 2 treatment are needed to induce complete male to female sex reversal in rainbow trout, tilapia and other teleosts (Krisfalusi & Nagler 2000, Bhandari et al 2005, Gennotte et al 2014. This might be explained by the following two possible reasons: i) though the ovarian differentiation occurs earlier than testicular differentiation, the male determining gene, such as amhy in tilapia, expresses even earlier than the female pathway genes (Eshel et al 2014, Li et al 2015a, and therefore high doses of E 2 are needed to antagonize the male pathway genes, which is further supported by the differential sensitivity of XY and YY genotypes to embryonic induced-feminization (Gennotte et al 2014); and ii) almost all steroidogenic enzyme genes are expressed in XX gonads but not in XY gonads at 5 dah (Tao et al 2013), and therefore high doses of E 2 are needed to induce those genes, especially cyp19a1a, expression for ovarian differentiation. Signals of Dmrt1 and Cyp19a1a could be simultaneously detected at 10 dah in the MT-XX gonad while Cyp19a1a disappeared latter on.…”

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].…”

“…In the Nile tilapia the 233-bp deleted amh gene, which was initially called amhy [Eshel et al, 2014] and later amhΔY [Li et al, 2015], was identified in males and considered to be specific for the Y chromosome and was always associated to the male sex determinant amhy in this species. We analyzed the existence of the amhΔY in different sexual genotypes of the domesticated Manzala strain using primers that covered both the deleted and undeleted regions of the amh genes producing a fragment of 800 bp and 1,000 bp.…”

“…Epigenetic marks other than methylation are only starting to be analyzed in fish during sex determination and differentiation. For instance microRNA, small non-coding RNAs that inhibit at post-transcriptional level target-gene expressions, have begun to be profiled between differentiating ovaries and testes in the Nile tilapia [Eshel et al, 2014;Tao et al, 2016]. Integration of miRNA and mRNA was compared for early differentiating XX and XY Nile tilapia identifying 130 novel miRNA of which 49 were unique for XX and 45 solely found in XY gonads [Tao et al, 2016].…”

“…The underlying molecular mechanisms modifying testis or ovarian thresholds leading to sex reversal in fish are slowly being better deciphered with transcriptome microarrays studies Eshel et al, 2014] or using next-generation sequencing techniques (NGS) [Yano et al, 2012;Shao et al, 2014;Tao et al, 2016] coupled to overexpression and knockdown studies [Imai et al, 2015;Li et al, 2015;Chakraborty et al, 2016]. The hormonal estrogen status has been known for a long time to be an important factor affecting the sex threshold towards ovarian differentiation via changes of aromatase, the estrogen-producing enzyme or its gene cyp19a1a [Guiguen et al, 1999[Guiguen et al, , 2010Kitano et al, 1999;D'Cotta et al, 2001aD'Cotta et al, , 2001b.…”