The differences between sexes and the concept of sex determination have always fascinated, yet troubled philosophers and scientists. Among the animals that reproduce sexually, teleost fishes show a very wide repertoire of reproductive modes. Except for the gonochoristic species, fish are the only vertebrates in which hermaphroditism appears naturally. Hermaphroditism refers to the capability of an organism to reproduce both as male and female in its life cycle and there are various forms of it. In sequential hermaphroditism, an individual begins as female first and then can change sex to become male (protogyny), or vice versa (protandry). The diverse sex-phenotypes of fish are regulated by a variety of sex determination mechanisms, along a continuum of environmental and heritable factors. The vast majority of sexually dimorphic traits result from the differential expression of genes that are present in both sexes. To date, studies regarding the sex-specific differences in gene expression have been conducted mainly in sex determination systems of model fish species that are well characterized at the genomic level, with distinguishable heteromorphic sex chromosomes, exhibiting genetic sex determination and gonochorism. Among teleosts, the Sparidae family is considered to be one of the most diversified families regarding its reproductive systems, and thus is a unique model for comparative studies to understand the molecular mechanisms underlying different sexual motifs. In this study, using RNA sequencing, we studied the transcriptome from gonads and brains of both sexes in five sparid species, representatives of four different reproductive styles. Specifically, we explored the sex-specific expression patterns of a gonochoristic species: the common dentex Dentex dentex, two protogynous hermaphrodites: the red porgy Pagrus pagrus and the common pandora Pagellus erythrinus, the rudimentary hermaphrodite sharpsnout seabream Diplodus puntazzo, and the protandrous gilthead seabream Sparus aurata. We found minor sex-related expression differences indicating a more homogeneous and sexually plastic brain, whereas there was a plethora of sex biased gene expression in the gonads. The functional divergence of the two gonadal types is reflected in their transcriptomic profiles, in terms of the number of genes differentially expressed, as well as the expression magnitude (i.e. fold-change differences). The observation of almost double the number of up-regulated genes in males compared to females indicates a male-biased expression tendency. Focusing on the pathways and genes implicated in sex determination/differentiation, we aimed to unveil the molecular pathways through which these non-model fish species develop a masculine or a feminine character. We observed the implicated pathways and major gene families (e.g. Wnt/b-catenin pathway and Retinoic-acid signaling pathway, Notch, TGFβ) behind sex-biased expression and the recruitment of known sex-related genes either to male or female type of gonads in these fish. (e.g Dmrt1, Sox9, Sox3, Cyp19a, Filgla, Ctnnb1, Gsdf9, Stra6 etc.). We also carefully investigated the presence of genes reported to be involved in sex determination/differentiation mechanisms in other vertebrates and fish and compared their expression patterns in the species under study. The expression profiling exposed known candidate molecular-players/genes establishing the common female (Cyp19a1, Sox3, Figla, Gdf9, Cyp26a, Ctnnb1, Dnmt1, Stra6) and male identity (Dmrt1, Sox9, Dnmt3aa, Rarb, Raraa, Hdac8, Tdrd7) of the gonad in these sparids. Additionally, we focused on those contributing to a species-specific manner either to female (Wnt4a, Dmrt2a, Foxl2 etc.) or to male (Amh, Dmrt3a, Cyp11b etc.) characters, and discussed the expression patterns of factors that belong to important pathways and/or gene families in the SD context, in our species gonadal transcriptomes. Taken together, most of the studied genes form part of the cascade of sex determination, differentiation, and reproduction across teleosts. In this study, we focused on genes that are active when sex is established (sex-maintainers), revealing the basic “gene-toolkit” & gene-networks underlying functional sex in these five sparids. Comparing related species with alternative reproductive styles, we saw different combinations of genes with conserved sex-linked roles and some “handy” molecular players, in a “partially- conserved” or “modulated” network formulating the male and female phenotype. The knowledge obtained in this study and tools developed during the process have set the groundwork for future experiments that can improve the sex control of this species and help the in-deep understanding the complex process of sex differentiation in the more flexible multi-component systems as these studied here.
Development and Application of Quantitative Gene Expression Assays in Wild-Caught, Non-Model Fish Species