Comparative piRNAs Profiles Give a Clue to Transgenerational Inheritance of Sex-Biased piRNAs in Cynoglossus semilaevis.

Piwi interacting RNAs (piRNAs) are involved in the epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, especially in spermatogenesis. There are many related reports on model organisms, such as flies and mice. In fish, however, there are few studies on piRNAs. Cynoglossus semilaevis, a benthic warm water flatfish, with remarkable sexual dimorphism, especially the "pseudo males" with sex reversal, mating with normal females to produce viable offspring, is an ideal material for the study of sex development. Here, sperm piwi-interacting RNAs profiles of Cynoglossus semilaevis were characterized, comparing between male and pseudomale groups. Differential piRNAs were identified with their predicted and annotated targets. Attention was then focused on candidate piRNAs associated with sex development and methylation. We continued to compare the expression levels of 10 candidates differentially expressed piRNAs in F1 spermatozoa. Quantitative RT-PCR demonstrated that five of the ten piRNAs showed sex bias consistent with parental sequencing results, with four significantly higher expression level in sperm of five males offspring than that of pseudomales, while one piRNAs showed the opposite expression profile. The five signature piRNAs (piR-mmu-49600337, piR-mmu-95849, piR-xtr-7474223, piR-xtr-1790334, and piR-mmu-4491546) could be employed as male-specific molecular biomarkers for C. semilaevis. Besides, this study also implied the possibility of transgenerational inheritance of sex-biased piRNAs exiting in sperm of Cynoglossus semilaevis.