Identification and application of piwi-interacting RNAs from seminal plasma exosome in Cynoglossus semilaevis
: Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and posttranscriptional 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 C. semilaevis because of its commercial
... 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, Cynoglossus semilaevis , were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in seminal plasma exosomes from males and pseudo-male 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 pseudo-males. 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 "pseudo-males" in C. semilaevis . Background Half smooth tongue sole (Cynoglossus semilaevis), a commercially valuable flatfish that is widely distributed in Chinese coastal waters, is commonly found in shallow waters on a muddy or sandy bottom 1 . Many previous studies have shown that C. semilaevis employs a heterogametic sex determination system (ZW/ZZ) 2,3 and has significant sexual dimorphism, with a larger female body size and faster growth rate 4,5 . This species is attracting more attention in reproductive and sexrelated research, and could become a tool to study sex determination in fish 6 . Furthermore, this species also exists as pseudo-male fish, both in nature and aquaculture 7 . The high proportion of 13 Declarations Ethics approval and consent to participate Weizhuo Ltd. (Tangshan, China) provided live C. semilaevis as semen donors for research. The methods for C. semilaevis research were carried out in accordance with the relevant guidelines and regulations. The protocols were approved by the academic ethics committee of Shanghai Ocean University Consent for publication Not applicable Availability of data and materials All sequence alignment and screened out data are presented in format in supplementary datasets 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11.