Expression and regulation of 42Sp50 in spotted scat (Scatophagus argus)

Abstract

42Sp50 is an isoform of the eukaryotic translation elongation factor 1 A (eEF1A) and is vital for fish ovarian development. Spotted scat (Scatophagus argus) is a popular marine cultured fish species in Southern Asia and China, and its artificial reproduction is complicated, with a relatively low success ratio in practice. In this study, the 42Sp50 gene was cloned from spotted scat. Tissue distribution analysis showed that 42Sp50 was mainly expressed in the ovary. qRT-PCR showed that 42Sp50 expression levels gradually decreased insignificantly in the ovaries from phase II to IV. Western blot analysis showed that 42Sp50 was highly expressed in the ovary, while it was almost undetectable in the testis. Immunohistochemistry analysis stained 42Sp50 mainly in the cytoplasm of the previtellogenic oocytes in ovaries of normal XX-female and sex-reversed XY-female. Aside from fish and amphibians, 42Sp50 was also identified in some reptile species using genomic database searching. Analyses of the transcriptome data from four different fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), and Hong Kong catfish (Clarias fuscus)) revealed ovaries biased expression of 42Sp50 in all, similar to spotted scat. While the neighbor genes of 42Sp50 did not show ovary biased expression in the fish species analyzed. Bisulfite Sequencing PCR (BSP) results showed that the DNA methylation level of 42Sp50 promoter was low in ovaries, testes, and muscles. The luciferase reporter assay demonstrated that Dmrt4 activated 42Sp50 expression in the presence of Sf1 or Foxh1. These results suggest that 42Sp50 may be involved in regulating the early phase oocytes development of spotted scat.

Preliminary Trial of Male to Female Sex Reversal by 17β-Estradiol in Combination with Trilostane in Spotted Scat (Scatophagus argus)

“…Our IHC results showed that the location of 42Sp50 in the ovaries of XY-completely sex-reversed individuals was very similar to that in the ovaries of XX females. The location of 42Sp50 in the XY sex-reversed fish obtained by E 2 treatment is also similar to that of the untreated XX females (Figure 5) [17]. Therefore, we speculate that the gonads of XY-completely sex-reversed spotted scat obtained through the preliminary combined treatment trial may have functional ovaries based on the ovarian gene expression profiles.…”

“…Genomic DNA (gDNA) was extracted using a DNA extraction kit (AD1173, Guangzhou Aitier Biotechnology Co., Ltd., Guangzhou, China). The gDNA was used to accurately identify the sex of the collected spotted scat using sex identification markers previously obtained in our laboratory [17]. Blood was collected from the caudal vein and kept at 4 • C overnight.…”

“…To localize Gsdf and 42Sp50, immunohistochemistry was performed on the gonads of treated spotted scat. The specific spotted scat Gsdf and 42Sp50 polyclonal antibodies were produced in our laboratory [17,19]. Antibody against Gsdf from goat and antibody against 42Sp50 from rabbit were both diluted to 1:500.…”