Brain and Pituitary Transcriptome Analyses Reveal the Differential Regulation of Reproduction-Related LncRNAs and mRNAs in Cynoglossus semilaevis

Long noncoding RNAs (lncRNAs) have been identified to be involved in half-smooth tongue sole (Cynoglossus semilaevis) reproduction. However, studies of their roles in reproduction have focused mainly on the ovary, and their expression patterns and potential roles in the brain and pituitary are unclear. Thus, to explore the mRNAs and lncRNAs that are closely associated with reproduction in the brain and pituitary, we collected tongue sole brain and pituitary tissues at three stages for RNA sequencing (RNA-seq), the 5,135 and 5,630 differentially expressed (DE) mRNAs and 378 and 532 DE lncRNAs were identified in the brain and pituitary, respectively. The RNA-seq results were verified by RT-qPCR. Moreover, enrichment analyses were performed to analyze the functions of DE mRNAs and lncRNAs. Interestingly, their involvement in pathways related to metabolism, signal transduction and endocrine signaling was revealed. LncRNA-target gene interaction networks were constructed based on antisense, cis and trans regulatory mechanisms. Moreover, we constructed competing endogenous RNA (ceRNA) networks. In summary, this study provides mRNA and lncRNA expression profiles in the brain and pituitary to understand the molecular mechanisms regulating tongue sole reproduction.

A Z-Linked E3 Ubiquitin Ligase Cs-rchy1 Is Involved in Gametogenesis in Chinese Tongue Sole, Cynoglossus semilaevis

Ubiquitin ligase (E3) plays a versatile role in gonadal development and spermatogenesis in mammals, while its function in fish is little reported. In this study, a Z-chromosome linked ubiquitin ligase rchy1 in C. semilaevis (Cs-rchy1) was cloned and characterized. The full-length cDNA was composed of 1962 bp, including 551 bp 5′UTR, 736 bp 3′UTR, and 675 bp ORF encoding a 224-amino-acid (aa) protein. Cs-rchy1 was examined among seven different tissues and found to be predominantly expressed in gonads. In testis, Cs-rchy1 could be detected from 40 days post hatching (dph) until 3 years post hatching (yph), but there was a significant increase at 6 months post hatching (mph). In comparison, the expression levels in ovary were rather stable among different developmental stages. In situ hybridization showed that Cs-rchy1 was mainly localized in germ cells, that is, spermatid and spermatozoa in testis and stage I, II and III oocytes in ovary. In vitro RNA interference found that Cs-rchy1 knockdown resulted in the decline of sox9 and igf1 in ovarian cell line and down-regulation of cyp19a in the testicular cell line. These data suggested that Cs-rchy1 might participate in gonadal differentiation and gametogenesis, via regulating steroid hormone synthesis.

Comprehensive CircRNA Profiling and Selection of Key CircRNAs Reveal the Potential Regulatory Roles of CircRNAs throughout Ovarian Development and Maturation in Cynoglossus semilaevis

CircRNAs are novel endogenous non-coding small RNAs involved in the regulation of multiple biological processes. However, little is known regarding circRNAs in ovarian development and maturation of fish. Our study, for the first time, provides the genome-wide overview of the types and relative abundances of circRNAs in tongue sole tissues during three ovarian developmental stages. We detected 6790 circRNAs in the brain, 5712 in the pituitary gland, 4937 in the ovary and 4160 in the liver. Some circRNAs exhibit tissue-specific expression, and qRT-PCR largely confirmed 6 differentially expressed (DE) circRNAs. Gene Ontology and KEGG pathway analyses of DE mRNAs were performed. Some DE circRNA parental genes were closely associated with biological processes in key signalling pathways and may play essential roles in ovarian development and maturation. We found that the selected circRNAs were involved in 10 pathways. RNase R digestion experiment and Sanger sequencing verified that the circRNA had a ring structure and was RNase R resistant. qRT-PCR results largely confirmed differential circRNA expression patterns from the RNA-seq data. These findings indicate that circRNAs are widespread in terms of present in production-related tissues of tongue sole with potentially important regulatory roles in ovarian development and maturation.