Expression of scp3 and dazl reveals the meiotic characteristics of the olive flounder Paralichthys olivaceus†.

Olive flounder Paralichthys olivaceus is an important cultured marine fish. We found that the meiosis marker scp3 and its intrinsic regulator dazl were mainly expressed in the gonads. During the ovarian differentiation, scp3 signal was detected first in pre-meiotic oogonia at 60-mm total length (TL) and then in primary oocytes at 80- and 100-mm TL, with a sharp increase in scp3 expression level observed at 80- and 100-mm TL. Dazl signal was detected in primordial germ cells at 30-mm TL and oogonia at 60-mm TL, but no significant change of expression was observed. During the testicular differentiation period, scp3 and dazl expression remained at low levels, and scp3 signal was weakly detected in spermatogonia at 80-mm TL, whereas dazl signal was not found. During the ovarian developmental stages, the highest expression levels of scp3 and dazl were detected at stages I and II, respectively, and strong signals of scp3 and dazl were detected in primary oocytes and oocytes at phases I and II. In the testis, the high expression of scp3 and dazl was detected at stages II-IV and II-III, respectively. Scp3 signal was weakly observed in pre-meiotic spermatogonia at stages I and II and strongly detected in primary spermatocytes at stages III-V. Dazl was detected in the nuclei of spermatogonia and spermatids at stages II-IV. Furthermore, scp3 expression in the ovary could be promoted by 17α-ethynylestradiol and tamoxifen, whereas dazl expression could be downregulated by tamoxifen.

Function of Foxl2 and Dmrt1 proteins during gonadal differentiation in the olive flounder Paralichthys olivaceus.

Study on fish sex differentiation is important both from academic and practical aspects. Foxl2 and Dmrt1 are important transcription factors that should be involved in fish gonadal differentiation, but there is still no direct evidence to clarify their protein functions. Olive flounder Paralichthys olivaceus, an important mariculture fish in China, Japan, and Korea, shows sex-dimorphic growth. In this study, the Foxl2 and Dmrt1 proteins were detected in granulosa cells of the ovary and Sertoli cells of the testis, respectively, showing significant sex-dimorphic expression patterns. Then, bioactive high-purity Foxl2 and Dmrt1 recombinant proteins were obtained in vitro. Furthermore, effects of the recombinant Foxl2 and Dmrt1 during gonadal differentiation period were evaluated by intraperitoneal injection in juvenile fish. Compared with the control group, the male rate in the Dmrt1 group increased from 0 % to 82 %, showing for the first time in fish that the recombinant Dmrt1 could alter the sex phenotype. In addition, transcription levels of cyp19a and its transcription factors also changed after the recombinant Foxl2 and Dmrt1 injection. These findings reveal that Foxl2 and Dmrt1 are vital regulators for fish gonadal differentiation by regulating cyp19a expression, and also provide a new approach for sex control in fish aquaculture.

Sexual dimorphism of DNA and histone methylation profiles in the gonads of the olive flounder Paralichthys olivaceus.

DNA methylation and histone methylation are two types of the most important epigenetic modifications. However, research on their differential expression in gonads of male and female fish is limited. In this study, we examined the characteristics of DNA methylation and tri-methylation of lysine 4 of histone H3 (H3K4me3) modification profiles in the gonads of the wild-type and meio-gynogenetic olive flounders Paralichthys olivaceus. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the global DNA methylation level was higher in the testis than in the ovary. Real-time quantitative PCR (qPCR) results indicated that maintenance DNA methyltransferase gene dnmt1 and de novo DNA methyltransferase gene dnmt3a are highly expressed in the ovary, while DNA demethyltransferase genes tets are highly expressed in the testis. The inconsistency of DNA methylation and methyltransferase genes in the gonads might associate with the differential distribution in the testis. 5-mC mainly located in the spermatids of the testis was shown with immunohistochemistry (IHC). Furtherly, dnmt3a and tets are mainly located in spermatocytes and oocytes with in situ hybridization (ISH) analysis. As for H3K4me3, total level is higher in the ovary detected with western blot assay. IHC results showed that the signals of H3K4me3 in Sertoli cells of the testis were stronger than those in spermatocytes and spermatids. Methyltransferase gene kmt2b and demethylase genes kdm5a and kdm5c also exhibit much higher expression in the testis with qPCR, and ISH stronger signals of kmt2b and kdm5s were detected in spermatocytes. These results implied that DNA methylation and H3K4me3 are involved in the flounder sex differences and gametogenesis.

Transcription profiles and fatty acid composition of the artificial induced triploid alevin in Atlantic salmon Salmo salar.

Triploid Atlantic salmon Salmo salar is commercially available and ideal means of achieving sterility in fish. The expression patterns of triploid parr and smolt were described before. However, little is known about the gene expression and fatty acid composition of triploid alevins at endogenous nutritional stage, which is an important period for their survival and development. In this study, the development of the diploid and triploid Atlantic salmon embryos and hatched alevins before feeding was compared, and the results showed that there was no obvious morphological difference between them. And then, the transcription profiles of the triploid and diploid alevins on 3, 23 and 33 days post hatching (dph) were investigated by using RNA-seq after the yolk sacs were removed. There were more differentially expressed genes (DEGs) on 3 dph (2925) than those on 23 (589) and 33 dph (606). Compared with the diploid groups, the up-regulated genes in the triploid groups at the three sampling times were 1189, 339 and 340, respectively, while 1736, 250 and 266 genes were down-regulated. There were 2088 and 5215 DEGs on 23 and 33 dph compared with on 3 dph in the diploid groups, while 6533 and 9340 DEGS on 23 and 33 dph compared with 3 dph in the triploid groups. The functional enrichment analysis showed that the DEGs were mainly enriched to digestion and metabolism function, especially on 33 dph when the larvae were about to feeding. Meanwhile, 15 out of 22 kinds of fatty acids of alevins on 33 dph showed significant differences (P < 0.05), in which all the fatty acids contents in the triploid were lower. The results indicated the specific nutritional requirement in the triploid was represented as early as yolk sac stage. The present study gave an important vision in triploid Atlantic salmon breeding.

Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning.