Study of genetic factors and temperature influence on sex determination and differentiation in turbot

Abstract

Sex, as intuitive and simple as it may seem to us, poses some of the most interesting and complex questions when studying life. Sex is an intrinsic characteristic of most eukaryote species which eventually has led to the appearance of two differentiated adult phenotypes or sexes, males and females. This distinction rules a huge part of our lives and is the origin of important evolutionary processes based on intra-sex competition or inter-sex conflict due to sexual antagonism. Furthermore, sex is an important character for a plethora of species involved in human activities, for example in aquaculture many fish species present sex size dimorphisms where one sex grows faster than the other, and so knowing how sex is determined in each species is of the outmost interest. Traditionally, sex determination has been considered a cascade process with a master gene at the top, but recent findings have suggested that, instead, it might be a network process where different genetic and environmental factors can alter gonad fate, which in turn would be connected with a huge number of different sex determination mechanisms in vertebrates, especially in poikiloterms. In this new view of sex, the different players involved in sex differentiation gain relevance and their study may help us understanding how the fate of the gonad is determined. In this work, we have studied sex differentiation in turbot, a flatfish with a marked sex dimorphism where females grow faster than males. This species presents genetic sex determination, but also temperature effects on sex ratios have been reported, which seem to be family-dependant. Our aim was to study sex differentiation in turbot to gain knowledge about how sex is determined in this species and also in a broader sense in fish. This work consists of expression studies in turbot gonads using two different techniques: real-time PCR and microarrays. First of all, the real time PCR technique was setup for gonad development studies in turbot. The different methods available for reference gene stability calculation and efficiency determination were assessed. Then, using this information we performed an extensive expression study on turbot sex differentiation ranging from undifferentiated to differentiated gonads at three different temperatures. We found that the first molecular signs of sex differentiation are observed at 90 days post fertilization and that three genes, cyp19a1a, amh and vasa, can be used to sex turbot at this stage. Furthermore, the expression of genes involved in germ cell development pointed towards their involvement in early sex differentiation and possibly sex determination. Temperature effects on sex differentiation were also assessed in this study. A higher proportion of females was obtained at cold temperatures and several genes showed temperature dependant expression changes. Finally, to complete our study, we also performed a microarray analysis in turbot gonad samples from undifferentiated individuals to male and female juveniles. Female gonads were found to be more different from undifferentiated gonads than those of males, requiring the regulation of a large number of genes and the involvement of different processes including epigenetic mechanisms. Furthermore, the involvement of known sex differentiation genes and previously unrelated genes in sex differentiation was observed. This study has widened our knowledge on sex differentiation in turbot in particular and in fish in general, helping to understand the role of many genes involved in sex differentiation across the whole vertebrate taxa and pointing towards other genes which have been connected with sex for the first time. Our data suggest that a network model might be more accurate to explain sex determination in turbot, where the environment can interact with genetic factors and modify gonad fate.