| The Scatophagus argus, also called spotted scat, is an species in Perciformes, Acanthuroidei, Scatophagidae. The spotted scat is famous as an ornamental and commercial fish. As the species can resistance diseases and has strong environmental adaptability, in coastal ponds and cages spotted scat have become an important species aquaculture for its economic value. Most of fry come from the wild fishing on account of goand development not synchronous and not mature artificial breeding technology. The phenomenon has a strong effect on the scale of farming. So having a study on gonad development genes not only give a reference for Reproductive physiology but also pave the way to artificial breeding technology. There is a lot of research on morphology, histology and sex differention related genes, but the goand development molecular mechanisms remain poorly to be understood. So cloning and analyzing Scatophagus argus gonad related genes will give light up gonad development molecular mechanisms.Based on the Scatophagus argus transcriptome database and sequences of the related species, ERs’ fragments were gaind by using primers. Using RACE technique, three ERs full length c DNA were cloned. Semiquantitative PCR and q RT-PCR were made use of revealing their expression levels in different organs and all stages of gonad development. The results were reported as follows:1) The full length ERα sequence was 2621 bp, containing a 5′ untranslated Regions(UTR)of 241 bp, 3′-UTR is 472 bp, It encods 635 amino acides(aa). The the protein weight is about 69.38 KD. Semiquantitative PCR, ERα is extensively expressed in all examined adult tissues. Higher expression was detected in liver, brain, pituitary and testis of female and male fish. RT-q PCR revealed that the expression level of ERα in the early development of ovary and the phase oocyte full of vitelline were higher in gonad, but expression levels obviously increased along with the development of testis.2) The ERβ1 was 2891 bp in full length, containing a 5′-UTRof 597 bp, 3′-UTR of 611 bp and an ORF encoding 560 aa. The estimated MW of the protein is 63.02 KD. Semi-quantitative PCR, ERβ1 is expressed in all adult tissues. Higher expression was observed in brain, pituitary and testis of female and male fish, while lower expression in female liver and lower in male liver.3) The full length sequence of ERβ2 was 2410 bp, which containing a 5′-UTR of 281 bp, 3′-UTR of 168 bp and an ORF encoding 657 aa. The estimated MW of the protein is 72.44 KD. The hightest expression of ERβ2 were found in testis and female and male liver. The ERβ2 expression gradually decreased along with the development of ovary, but it stay high expression all the time in testis except in Spermiogenesis with little lower expreesion.Using ISH(in situ hybridization) with DIG antisense RNA probe detected ERβs distribution during gametogenesis. The results are as followed:1) ERβ1 was detected in all stages of ovary, such as oogonia, primary oocyte and second oocyte and mature oocyte, but mainly expressed in the cytoplasm of these oocyte. Highly expression were found along with spermatogenesis, but there was weak signals in primary spermatocyte, secondary spermatocyte and spermatid, but in leydig cell they have strong signals all the time.2) The ERβ2 expression signals were mainly found in oogonia. The strong sigals was detected in the cytoplasm of oocyte while expressed in the nucleus of oocyte that accumulating vitellin. ERβ2 was also expressed in leydig cells and germ cells.All in all, cloning and analyzing Scatophagus argus estrogen receptors expression in gonad development showed that these genes may have significant roles in ovary and testis development. ISH results showed that ERβs may be involved in gametogenesis, but they may have different molecular mechanismes. These results may pave the way to elucidate the Scatophagus argus reproductive molecular mechanism. |
PDF Full Text Request