Identification and application of piwi-interacting RNAs from seminal plasma exosomes in Cynoglossus semilaevis.

Background: Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Exosomes are important mediators of vesicle transport, and the piRNAs in exosomes might play an important role in cell communication and signal pathway regulation. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. We used Cynoglossus semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed "pseudomales" who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring.

Results: To determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, C. semilaevis, were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in exosomes of seminal plasma from males and pseudomale C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylation-related and transposition-related piRNAs. Their expression profiles were verified using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudomales. The signature piRNAs could be exploited as male-specific biomarkers in this fish.

Conclusions: These signatures provide an effective tool to explore the regulatory mechanism of sex development in C. semilaevis and may provide guidance for future research on the function of piRNAs in the generative mechanism of sex reversed "pseudomales" in C. semilaevis.