DNA methylation level of cyp19a1a and Foxl2 gene related to their expression patterns and reproduction traits during ovary development stages of Japanese flounder (Paralichthys olivaceus).

Foxl2 and cyp19a1a genes are crucial for the ovarian development, and Foxl2 could play a direct regulatory role on cyp19a1a transcription. In this study, we aimed to study DNA methylation status and mRNA expression patterns of Foxl2 and cyp19a1a genes during ovarian development of female Japanese flounder. The relative expression level of cyp19a1a and Foxl2 gene during the gonadal development stages was measured by quantitative PCR. Moreover, DNA methylation status in the promoter and coding regions of the two genes was detected by bisulfite sequencing. The estradiol-17ß (E2) was measured by radioimmunoassay. The results showed low expression levels of cyp19a1a and Foxl2 genes in stages II and V, while the highest expression levels were detected in stage IV. The variation trend of the methylation level of all CpG sites in promoter and exon 1 of cyp19a1a gene and three CpG rich regions in coding region of Foxl2 gene was negatively associated with their expression levels during the ovarian development. In addition, two CpG sites in promoter and seven CpG sites in exon 1 of cyp19a1a were on the putative transcription factors binding sequence. Further studies showed that the forkhead domain, which is important for Foxl2 binding to cyp19a1a was located in the F1 and F2 region. These results provide a powerful theoretical basis for the regulatory mechanism on Foxl2 regulating cyp19a1a and promoting gonadal differentiation towards the female pathway, and further reveal that Foxl2 and cyp19a1a play a vital role in the female Japanese flounder gonad development.

Genetic polymorphisms and DNA methylation in exon 1 CpG-rich regions of PACAP gene and its effect on mRNA expression and growth traits in half smooth tongue sole (Cynoglossus semilaevis).

The pituitary adenylate cyclase activating polypeptide (PACAP) is a new type of hypophysiotropic hormone and plays an important role in regulating the synthesis and secretion of growth hormone and gonadotropin. The research on the relationship between PACAP and different growth traits would contribute to explain its function during the process of growth. Moreover, epigenetic modifications, especially DNA methylation at the CpG sites of the SNPs, play important roles in regulating gene expression. The results suggest that a SNP mutation (c.C151G) in the PACAP gene of male half smooth tongue sole (Cynoglossus semilaevis) is significantly associated with growth traits and serum physiological and biochemical parameters such as inorganic phosphorus (P < 0.05). The SNP is located in a CpG-rich region of exon 1. Intriguingly, the transition (C→G) added a new methylation site of PACAP gene. This SNP was also significantly related to the expression and methylation level of PACAP (P < 0.05). Individuals with GG genotype had faster growth rates than those of CG and CC genotypes. Moreover, GG genotype had significantly higher PACAP expression level and lower methylation level than CG and CC genotypes. In the serum indexes, only inorganic phosphorus content within GG genotypes was significantly higher than CC genotypes. This implied that the mutation and methylation status of PACAP gene could influence growth traits and this locus could be considered as a candidate genetic or epigenetic marker for Cynoglossus semilaevis molecular breeding.