Transcriptomic Analysis Reveals Functional Interaction of mRNA–lncRNA–miRNA in Steroidogenesis and Spermatogenesis of Gynogenetic Japanese Flounder (Paralichthys olivaceus)

SIMPLE SUMMARY: Japanese flounder (Paralichthys olivaceus) is an important marine cultured fish that has a male heterogametic sex determination and shows significant sexual dimorphism with bigger females. Therefore, gynogenesis has presented great potential in breeding of all-female P. olivaceus. Non-coding RNAs are emerging as critical regulators of reproduction, and in this study, lncRNA–miRNA–mRNA interactions were investigated using high-throughput sequencing to reveal their roles in gynogenetic female and sex-reversed neo-male P. olivaceus. A considerable amount of steroidogenesis and spermatogenesis-related genes and ncRNAs were found, which may be regulated by let-7/miR-125b clusters and have significant functions during spermatogenesis of the neo-male P. olivaceus. These highly conserved and putatively interacted RNAs could replenish the genomic information of P. olivaceus and pave the way to understand the molecular mechanisms of gynogenesis effect and sex reversal in sexual development and gametogenesis of teleost fishes. ABSTRACT: Teleost fishes exhibit extraordinary diversity, plasticity and adaptability with their sex determination and sexual development, and there is growing evidence that non-coding RNAs (ncRNAs) are emerging as critical regulators of reproduction. Japanese flounder (Paralichthys olivaceus) is an important marine cultured fish that presents significant sexual dimorphism with bigger females, in which gynogenesis has been applied for aquaculture industry. In order to reveal the regulatory mechanisms of sexual development in gynogenetic female and sex-reversed neo-male P. olivaceus, the lncRNA–miRNA–mRNA interactions were investigated using high-throughput sequencing. A total of 6772 differentially expressed mRNAs (DEmRNAs), 2284 DElncRNAs, and 244 DEmiRNAs were obtained between gynogenetic female ovaries and sex-reversed neo-male testes. Genes in the steroid hormone biosynthesis and secretion pathway were enriched and mostly significantly upregulated in neo-male testes. Subsequently, network analysis uncovered high functional specificity for gynogenetic P. olivaceus sperm motility, as co-expressed DEmRNAs were significantly enriched in microtubule and cytoskeleton-related biological processes. Clustered miRNAs were characterized in the P. olivaceus genome with examples of the largest conserved let-7 clusters. The 20 let-7 members are distributed in 11 clusters and may not transcribe together with their neighboring miR-125b, with let-7 repressing cyp11a and miR-125b repressing esr2b, both as key steroidogenesis pathway genes. In summary, this study provides comprehensive insights into the mRNA–miRNA–lncRNA functional crosstalk in teleost sexual development and gametogenesis and will expand our understanding of ncRNA biology in teleost gynogenesis.