CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

Molecular and Physiological Effects of 17α-methyltestosterone on Sex Differentiation of Black Rockfish, Sebastes schlegelii.

It is widely known that all-female fish production holds economic value for aquaculture. Sebastes schlegelii, a preeminent economic species, exhibits a sex dimorphism, with females surpassing males in growth. In this regard, achieving all-female black rockfish production could significantly enhance breeding profitability. In this study, we utilized the widely used male sex-regulating hormone, 17α-methyltestosterone (MT) at three different concentrations (20, 40, and 60 ppm), to produce pseudomales of S. schlegelii for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of S. schlegelii, while short term had no significant impact. Histological analysis confirmed sex reversal at all MT concentrations; however, both medium and higher MT concentrations impaired testis development. MT also influenced sex steroid hormone levels in pseudomales, suppressing E2 while increasing T and 11-KT levels. In addition, a transcriptome analysis revealed that MT down-regulated ovarian-related genes (cyp19a1a and foxl2) while up-regulating male-related genes (amh) in pseudomales. Furthermore, MT modulated the TGF-ß signaling and steroid hormone biosynthesis pathways, indicating its crucial role in S. schlegelii sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in S. schlegelii and offers an initial insight into the underlying mechanism of sexual reversal in this species.

Transcriptome Analysis Revealed the Early Heat Stress Response in the Brain of Chinese Tongue Sole (Cynoglossus semilaevis).

As a common influencing factor in the environment, temperature greatly influences the fish that live in the water all their life. The essential economic fish Chinese tongue sole (Cynoglossus semilaevis), a benthic fish, will experience both physiological and behavioral changes due to increases in temperature. The brain, as the central hub of fish and a crucial regulatory organ, is particularly sensitive to temperature changes and will be affected. However, previous research has mainly concentrated on the impact of temperature on the gonads of C. semilaevis. Instead, our study examines the brain using transcriptomics to investigate specific genes and pathways that can quickly respond to temperature changes. The fish were subjected to various periods of heat stress (1 h, 2 h, 3 h, and 5 h) before extracting the brain for transcriptome analysis. After conducting transcriptomic analyses, we identified distinct genes and pathways in males and females. The pathways were mainly related to cortisol synthesis and secretion, neuroactive ligand-receptor interactions, TGF beta signaling pathway, and JAK/STAT signaling pathway, while the genes included the HSP family, tshr, c-fos, c-jun, cxcr4, camk2b, and igf2. Our study offers valuable insights into the regulation mechanisms of the brain's response to temperature stress.