Gene expression analysis at the onset of sex differentiation in turbot (Scophthalmus maximus)

Robledo et al. BMC Genomics (2015) 16:973 DOI 10.1186/s12864-015-2142-8 RESEARCH ARTICLE Open Access Gene expression analysis at the onset of sex differentiation in turbot (Scophthalmus maximus) Diego Robledo1, Laia Ribas2, Rosa Cal3, Laura Sánchez4, Francesc Piferrer2, Paulino Martínez4 and Ana Viñas1* Abstract Background: Controlling sex ratios is essential for the aquaculture industry, especially in those species with sex dimorphism for relevant productive traits, hence the importance of knowing how the sexual phenotype is established in fish. Turbot, a very important fish for the aquaculture industry in Europe, shows one of the largest sexual growth dimorphisms amongst marine cultured species, being all-female stocks a desirable goal for the industry. Although important knowledge has been achieved on the genetic basis of sex determination (SD) in this species, the master SD gene remains unknown and precise information on gene expression at the critical stage of sex differentiation is lacking. In the present work, we examined the expression profiles of 29 relevant genes related to sex differentiation, from 60 up to 135 days post fertilization (dpf), when gonads are differentiating. We also considered the influence of three temperature regimes on sex differentiation. Results: The first sex-related differences in molecular markers could be observed at 90 days post fertilization (dpf) and so we have called that time the onset of sex differentiation. Three genes were the first to show differential expression between males and females and also allowed us to sex turbot accurately at the onset of sex differentiation (90 dpf): cyp19a1a, amh and vasa. The expression of genes related to primordial germ cells (vasa, gsdf, tdrd1) started to increase between 75–90 dpf and vasa and tdrd1 later presented higher expression in females (90-105 dpf). Two genes placed on the SD region of turbot (sox2, fxr1) did not show any expression pattern suggestive of a sex determining function. We also detected changes in the expression levels of several genes (ctnnb1, cyp11a, dmrt2 or sox6) depending on culture temperature. Conclusion: Our results enabled us to identify the first sex-associated genetic cues (cyp19a1a, vasa and amh) at the initial stages of gonad development in turbot (90 dpf) and to accurately sex turbot at this age, establishing the correspondence between gene expression profiles and histological sex. Furthermore, we profiled several genes involved in sex differentiation and found specific temperature effects on their expression. Keywords: Fish, Gonad, Development, qPCR, Genes, Sex ratio, Aromatase, Male, Female, Temperature Background Sex was thought to have arisen in a single evolutive event in the last common ancestor of all eukaryotes, since sexual reproduction is almost universal and exclusive of this group [1]. Considering its consequences over the lifespan of an organism and its influence on population demography, it is thought that the sex determination (SD) mechanism should be under strong selection forces * Correspondence: 1 Departamento de Genética, Facultad de Biología, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Full list of author information is available at the end of the article [2]. However, sex can be established by many different and fast-evolving mechanisms [3, 4], indicating that SD triggers have emerged several times throughout evolution [5]. Within vertebrates, different sex determining systems have been described. In therian mammals, with a XX/XY chromosome system, sex depends on the presence of the Sry gene, a paralogue of SOX3, on the Y chromosome [6], while in birds (chicken) with a ZZ/ZW chromosome system, the DMRT1 gene with a double dosage is required for testis development [7]. Also, in Xenopus laevis the DM-W gene, a paralogue of DMRT1, is responsible for SD [8]. When dmY, belonging to the DM family of © 2015 Robledo et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Robledo et al. BMC Genomics (2015) 16:973 transcription factors like DMRT1 and DM-W, was found to be the sex determining gene (SDG) of the fish Oryzias latipes, a biased and recurrent recruitment of specific SDGs or families throughout evolution was suggested [9]. However, later findings in fish do not seem to support this hypothesis. Fish, with around 30,000 species [10], is the most diverse group of vertebrates and its study has broadened our knowledge on SD. Fish diversity is also reflected by the variety of reproductive strategies: unisexuality, different types of hermaphroditism and gonochorism; and also by the diversity of SD mechanisms [11]. In the last years, an important effort has been made in order to identify the SDG in several model and aquaculture fish species. Different productive traits are sex-associated in farm fish such as growth rate, color, taste and flesh quality; hence, the interest of the industry in producing monosex stocks [12]. Detailed information at gene level is available for only a limited number of fish species. Five different master SDGs have been identified so far: dmY /dmrt1by in Oryzias latipes and in O. curvinotus [13], gsdf in O. luzonensis [14], amhy in Odontesthes hatchery [15], amhr2 in Takifugu rubripes, T. pardalis and T. poecilonotus [16], and sdY in salmonid family [17]. Recently, a distant cis-regulatory element of Sox3 necessary for male determination in O. dancena, a species with a XX/ XY SD system, has also been identified [18], and dmrt1 has been suggested as the SDG in Cynoglossus semilaevis [19]. However, little information is available, not only on the SDG, but also on the initial molecular pathways related to sexual differentiation. Traditionally, SD has been related to the switching mechanism of a hierarchical genetic network that causes the activation of downstream genes involved in gonad differentiation (GD) leading to the differentiation of testes or ovaries [20]. Thus, concerning whether the first difference between sexes is the expression of a gene/s or the strength of an environmental factor, SD can be genetic or environmental, although both ways can coexist [12, 21]. In the classical view of SD and GD, the downstream genes of the cascade were assumed to be highly conserved, and only the genes at the top of the cascade would change by gene duplication (and by the recruitment of a downstream gene) or by allelic diversification, establishing a new SD (...truncated)