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

Simple SummaryThe sexual growth dimorphism prevails in animals and this phenomenon is even more obvious in marine fish, so understanding the mechanism of gonadal development and gametogenesis is of great importance for sex control, thus increased productivity in aquaculture. In mammal, ubiquitin ligase plays a versatile role in gonadal development and spermatogenesis, whereas its function in fish is little reported. Using Cynoglossus semilaevis (one-year-old female individual usually grows 2-4 times bigger than male) as the fish model, a Z-chromosome linked ubiquitin ligase neurl3 was previously identified and characterized, which suggested its involvement in spermatogenesis. However, in this study, characterization of another Z-chromosome linked ubiquitin ligase Cs-rchy1 suggested it might function both in spermatogenesis and oogenesis, as well as the potential role in growth. These data may provide the genetic resource for gene editing or marker exploration in future.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 & PRIME;UTR, 736 bp 3 & PRIME;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.