Microarray technology is a powerful tool for studying genome-wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next-generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A-SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A-SG than in testicular somatic cells whereas supporting-cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A-SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher-levels in A-SG than somatic cells, by quantitative RT-PCR. In addition, three transcripts analyzed with in situ hybridization showed A-SG-specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A-SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout.

Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24 h. Among them, 11-ketotestosterone at 100 ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17β-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.