Role and evolution of a potential Sex Determining gene in Salmonids. – SDS

Our results allowed the identification and characterization of the master sex determining gene in rainbow trout,Oncorhynchus mykiss. This gene,sdY(«sexually dimorphic on the Y chromosome»), is only found on the Y chromosome of the males, and is the first gene to be expressed specifically in the male undifferentiated gonad in rainbow trout embryos. Using a targeted inactivation strategy we created sdY knockout genetic males (XY) in rainbow trout that turned into phenotypic females. In contrast, the overexpression of sdYin genetic females (XX) produced phenotypic males. As this gene is necessary and sufficient for testicular differentiation it fulfills all the required characteristics to be the master sex determining gene in trout. The sdY gene is then a new sex determining gene in the very short list of known sex determinants in vertebrates.

A very strong originality of sdYis its homology, as in contrast to other known sex determining genes that are the result of the duplication of known genes in the gonadal sex differentiation cascade, the sdY gene encodes for a protein involved in the immune response regulation. This could suggest that the immune response regulation pathway could be important for gonadal sex differentiation or that sdY evolved a totally new function

As this gene is also conserved as a male-specific sequence in many salmonid species, this can now allow the development of molecular sexing strategies (2). Such a molecular sexing tool should be especially useful in the monitoring and conservation of wild salmonid populations (like for instance in Atlantic salmon or in Brown trout in Europe), and also in the development of better sex control techniques in aquaculture. It could be also used for the aquatic environmental quality survey allowing the identification of masculinization or feminization induced by pollutants in water and their impact in wild population of salmonids.

Yano A, Guyomard R, Nicol B, Jouanno E, Quillet E, Klopp C, Cabau C, Bouchez O, Fostier A, Guiguen Y. 2012. An immune-related gene evolved into the master sex-determining gene in rainbow trout, Oncorhynchus mykiss. Current Biology. 22(15):1423-8.
Yano A, Nicol B, Jouanno E, Quillet E, Fostier A, Guyomard R, Guiguen Y. 2013. The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids. Evolutionary Applications, 6(3):486-96.

Fish exhibits a very large diversity of sex determination systems including species with either environmental or genetic sex-determination. In species with genetic (monofactorial) sex determination, master sex determining genes lying on sex chromosome, drive the gonadal differentiation process by switching on a developmental program that ultimately leads to testicular and ovarian differentiation. In fish, despite very important research efforts, only one master sex determining gene, called dmrt1Y, has been characterized in the Japanese medaka, Oryzias latipes; but this master gene has been shown to be restricted to some species of the genus Oryzias. This lack of knowledge on master sex determining genes in fish may be explained by the hypothesis that fish sex chromosomes are generally considered as young chromosomes and that these new sex chromosomes have emerged independently in different fish lineages, leading to a large diversity of master sex regulators. In salmonids, an important family which includes the rainbow trout, one of the most studied fish, the sex determination system is XX/XY. Two partners of the present project have recently identified a gene called SDY for “Sexually Dimorphic on the Y” that is specifically expressed in embryonic male gonads and completely Y-linked in males of rainbow trout and brown trout which belong to distinct salmonid genera. These results strongly support the hypothesis that SDY is the sex determinant master gene in these two species and possibly in other salmonids. This project has then two major objectives : (1) to characterize the function of SDY including whether it is the master sex determinant gene in rainbow trout and (2) to study its genomics and functional evolution across the salmonid family. To address the first question, the gonadal differentiation in rainbow trout will be analyzed in SDY Knockout and SDY over-expressing animals. To further characterize the biological function of SDY and its mechanisms of action, in vitro investigations will be carried out using Yeast double hybrid screening for protein binding partners and some biochemical characterizations of the SDY protein. Gene expression studies will be also performed in SDY transfected cell lines to identify downstream genes of the SDY pathway. To answer the second question on the evolution of SDY, SDY homologous cDNA will be first searched in grayling and European whitefish, two salmonids evolutionary distant from rainbow trout and brown trout, using NGS sequencing of transcripts in differentiating gonads. This approach will be also carried out in Northern pike, a non salmonid fish that will be studied as an outgroup species in order to search whether there is a SDY homolog outside the salmonid family. In grayling and whitefish, the linkage groups harboring the sex locus and SDY will be mapped. BAC libraries for grayling and whitefish will be screened for homologous SDY sequences that will be used as probes to characterize the evolution of sex chromosomes using FISH and sequenced to compare the SDY locus sequence across salmonids. The successful characterization of a master sex determining gene in rainbow trout will be of major importance taking into account the very low number of species in which such genes have been characterized in vertebrates. From a comparative point of view this project will also have a great impact on what is already known on the evolution of genetic sex determination in vertebrates in general and more specifically in salmonids species, in which the question about the uniqueness of such a master sex determining gene is still a matter of debate. This characterization of a conserved master sex-determining gene in salmonids will also have important practical outcomes for molecular sexing that will be important for a better control of sex determination in aquaculture and for ecology and ecotoxicology research in species that are both economically and environmentally very important.