Characterization of Novel Testis Specific Genes During Sex Differentiation in Rainbow Trout (Oncorhynchus mykiss) Using a Next Generation Sequencing (NGS) Approach.

Abstract

Fish displays a large diversity of sexual development systems including genetic and environmental sex determination or sex reversal influenced by social influence. Although sex determination and sex differentiation in fish are interesting processes, knowledge on their molecular mechanisms is still limited. In order to better understand these processes, we used a NGS approach to characterize in detail gene expression profiles during early gonadal sex differentiation using rainbow trout as a fish model. Rainbow trout sex determination is strictly genetic with a male heterogametic system (XX/XY). Sex differentiation is however strongly influenced by steroids, allowing the production of sex inversed male breeders with a XX or YY genotypes. These special male breeders (XX or YY) mated with normal females (XX) produce all-female (XX) and all-male (XY) genetic populations that are a key biological resource to study early steps of gonadal differentiation. In addition, rainbow trout embryos have a relatively large body size and a slow development that makes possible to sample embryonic gonads at the very beginning of the gonadal development process (around hatching i.e., 35 days post fertilization [dpf]). Doubled-strand cDNA libraries were constructed from pooled testis and pooled ovaries sampled in 35 dpf rainbow trout embryos and these libraries were sequenced using the Roche 454 Titanium technology. This sequencing yielded 350,638 reads from the ovary library and 349,465 reads from the testis library. All these sequences have been assembled and mapped onto the existing rainbow trout transcriptome resulting in the identification of 42,044 "transcripts" (bioinformatics contigs) expressed in differentiating gonads. To characterize genes with sex dimorphic expression pattren, we selected transcripts containing more than 30 total reads with at least a 2 fold change between sexes. 48 transcripts were selected from the testis and 74 transcripts were selected from the ovary. Some of these transcripts were further characterized during early gonadal differentiation (from 35-70 dpf) using qPCR. Among those transcripts, we identified one previously uncharacterized gene with specific expression in the testis at very early stages of gonadal development, with no expression detected in the female differentiating ovaries. Whole mount in situ hybridization (WISH) using male and female embryos (from 28-70 dpf) showed very restricted expression pattern in some epithelial and somatic peri-germinal cells of the differentiating testis. The characterization of such novel testis specific genes during early gonadal differentiation demonstrates the interest of characterizing gene expression profiles during early gonadal sex differentiation using NGS approach. Therefore, analyzing more of this subset of sequence information will lead to a better knowledge of the molecular mechanisms of sex determination and sex differentiation in fish. (poster)