Transcriptional analysis of testis maturation using trout cDNA macroarrays

The project seeks to identify genes involved in key stages of trout spermatogenesis and their regulation. Within the framework of the French project of farm animal genomics (AGENAE) we produced an original normalised trout testis cDNA library and obtained 1152 trout ESTs corresponding to 967 potential genes. To study the expression of those genes throughout first stages of spermatogenesis, we used nylon macroarray. Gonads in stage of immaturity (stage I), or at initiation of spermatogonial proliferation (stage II), meiosis (stage III) or spermiogenesis were selected by histological analysis. Total RNA was extracted and then used to produce complex targets labelled with [33P]dCTP and hybridised with cDNA arrays. After filtering and normalisation of hybridisation signals, genes presenting differential expression as revealed by ANOVA analysis were submitted to k-means clustering and hierarchical classification. Genes were separated into five clusters which presented distinct profiles. One cluster overexpressed in stage I could be involved in the initial events of spermatogenesis as seminiferous tubule organisation. The second cluster displays a transient increase at the beginning of testicular recrudescence (stage II). Three other clusters group several genes involved in cell proliferation and protein synthesis and modification. One is particularly down-expressed during stage I, the two others show increased expression during stages III and IV and appear to be involved in spermatogonial and meiotic proliferation and in protein metabolism linked to cellular growth. This allows us to plan further experiments to better understand the functional implication of some of the genes that are found to be significantly regulated like CDC2, hematological and neurological expressed gene 1-like protein, HCDI protein, Mago Nashi, a BMP-like, and a steroid receptor binding protein. These data demonstrate the applicability of the array based technology using our trout cDNA arrays and highlight genes that are potential targets for the control of puberty and fertility in farmed fish.