Characterization and expression of androgen receptors in olive flounder.

Androgens are critical hormones that regulate sex differentiation, sexual maturation, and spermatogenesis in vertebrates, which is mainly mediated by androgen receptors (ARs). Reports on transcript variants of ar (AR encoding gene) in human are almost always associated with cancers and androgen insensitivity syndrome. However, the knowledge of ar variants in teleosts is scarce. In this study, arß and two transcript variants of arα (arα1 and arα2) in olive flounder (Paralichthys olivaceus) were cloned and analyzed. Their expression patterns were investigated in 16 adult female and male tissues by RT-PCR, respectively. arα1 was expressed in the majority of tissues excluding male liver, medulla oblongata and female cerebellum, with higher levels in male gonad, kidney, head kidney, intestine, stomach, spleen, heart and gill than in female. arα2 had similar expression patterns as arα1, with lower levels in general. arß was also widely expressed in various tissues excluding male spleen, female spleen and gill, with higher levels in male gonad, kidney, head kidney, intestine and lower levels in hypothalamus than in female. Compared with arß, much lower expression levels of arα1 and arα2 were detected in different brain areas. The real-time quantitative PCR (qPCR) results showed that the total arα expression level was relatively higher during olive flounder gonadal differentiation and before the onset of testis differentiation, whereas arß was expressed significantly higher during male gonadal differentiation period than female gonadal differentiation period. The in vitro transient transfection assays showed that ARα1, ARα2 and ARß could all suppress the activity of cyp19a (p450arom aromatase gene) promoter, and the inhibitory effect of ARα1 was dose dependent. Our results imply that arα1, arα2 and arß are sex-related genes and they might play important roles in gonadal differentiation in flounder.

Characterization and expression of StAR2a and StAR2b in the olive flounder Paralichthys olivaceus.

Steroidogenic acute regulatory protein 2 (StAR2) is a key protein in transporting cholesterol from the outer mitochondria membrane to the inner mitochondria membrane for sex steroid synthesis. In this study, two StAR2 gene isoforms, StAR2a and StAR2b, were isolated from the olive flounder Paralichthys olivaceus gonads. Semi-quantitative RT-PCR results indicated that their expression levels were higher in testis than those in ovary. StAR2a was mainly expressed in the thecal cells and ooplasm of ovary, and Leydig cells and spermatid of testis according to the results of in situ hybridization. The quantitative real-time PCR results showed that the expressions of StAR2a and StAR2b were high in undifferentiation gonads and differentiating testis, and then decreased in differentiated testis in the high temperature (28°C) and exogenous testosterone treatment groups. While, in the exogenous 17ß-estradiol treatment group, both genes' expression levels were high in differentiating ovary, and then significantly decreased in differentiated ovary (P<0.05). StAR2a and StAR2b expression levels were significantly down-regulated in the cultured testis cells treated with the 75 and 150µM cAMP, but significantly up-regulated in the cultured testis cells treated with the 300µM cAMP (P<0.05). Moreover, their expression levels were significantly up-regulated by transfecting the cultured testis cells with pcDNA3.1-NR5a2 and pcDNA3.1-NR0b1 (P<0.05). Above study showed that expression of StAR2 was regulated by cAMP and the transcription factors, NR5a2 and NR0b1, indicating that StAR2 may have functions in flounder gonadal differentiation and maintenance.

Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus).

WNT4 (wingless-type MMTV integration site family, member 4) is regarded as a key regulator of gonad differentiation in mammalians. However, the potential role of wnt4 in teleosts during gonad differentiation and development is still unclear. The full-length cDNA sequence of wnt4 in olive flounder (Paralichthys olivaceus) was obtained using RACE (rapid amplification of cDNA ends) technique. The wnt4 ORF contains 1059 nucleotides, encoding a protein with a signal peptide domain and a wnt family domain. Expression in tissues of adult flounders was analyzed by real-time RT-PCR. The results showed that wnt4 was widely expressed in multiple tissues of flounders, and the expression level was significantly higher in ovary than in testis. Then wnt4 expression pattern was investigated during gonadal differentiation period and at gonadal development stages (I-V). The results showed the expression levels were significantly higher in testis than in ovary during gonadal differentiation. Notably, wnt4 expression had a very significant increase before testis differentiation. At gonad different developmental stages, there was no expression signal at stage I or stage II, and the expression of wnt4 was much stronger in ovary than in testis at stage III and stage IV, followed by a faint expression in stage V in both sexes. Our results imply that cloned wnt4 could be wnt4a. It is a sex-related gene and its expression pattern in gonadal differentiation period of flounder is different from that in mammalians or other teleosts. Flounder wnt4 might play more important role in testis than in ovary during gonadal differentiation.

Molecular cloning and sexually dimorphic expression patterns of nr0b1 and nr5a2 in olive flounder, Paralichthys olivaceus.

The nr0b1 and nr5a2 genes, members of the nuclear receptor superfamily, are strong candidate genes involved in gonadal differentiation in several vertebrate species. In this study, an nr0b1 complementary DNA (cDNA) of 1446 bp, which encodes a predicted 298 amino acid protein, and an nr5a2 cDNA of 2425 bp, which encodes a deduced 523 amino acid protein, were obtained from olive flounder Paralichthys olivaceus. Both genes were expressed in multiple organ tissues of adult flounder, with a higher expression in ovary than in testis. Quantitative real-time RT-PCR was performed to investigate their temporal expression profiles in gonads during differentiation and at five development stages. Results indicated that nr0b1 and nr5a2 were expressed in primitive gonad and in the ensuing gonadal differentiation periods. In general, both genes were more highly expressed in ovary than in testis at all observed development stages. The expression level of cyp19a correlated with the nr5a2/nr0b1 ratio over the course of flounder gonadal differentiation; hence, nr0b1 and nr5a2 genes may be involved in flounder ovarian differentiation by regulating the expression of cyp19a.

Transcriptional responses of olive flounder (Paralichthys olivaceus) to low temperature.

The olive flounder (Paralichthys olivaceus) is an economically important flatfish in marine aquaculture with a broad thermal tolerance ranging from 14 to 23°C. Cold-tolerant flounder that can survive during the winter season at a temperature of less than 14°C might facilitate the understanding of the mechanisms underlying the response to cold stress. In this study, the transcriptional response of flounder to cold stress (0.7±0.05°C) was characterized using RNA sequencing. Transcriptome sequencing was performed using the Illumina MiSeq platform for the cold-tolerant (CT) group, which survived under the cold stress; the cold-sensitive (CS) group, which could barely survive at the low temperature; and control group, which was not subjected to cold treatment. In all, 29,021 unigenes were generated. Compared with the unigene expression profile of the control group, 410 unigenes were up-regulated and 255 unigenes were down-regulated in the CT group, whereas 593 unigenes were up-regulated and 289 unigenes were down-regulated in the CS group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that signal transduction, lipid metabolism, digestive system, and signaling molecules and interaction were the most highly enriched pathways for the genes that were differentially expressed under cold stress. All these pathways could be assigned to the following four biological functions for flounder that can survive under cold stress: signal response to cold stress, cell repair/regeneration, energy production, and cell membrane construction and fluidity.

Gonadal transcriptome analysis of male and female olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is an important commercially cultured marine flatfish in China, Korea, and Japan, of which female grows faster than male. In order to explore the molecular mechanism of flounder sex determination and development, we used RNA-seq technology to investigate transcriptomes of flounder gonads. This produced 22,253,217 and 19,777,841 qualified reads from ovary and testes, which were jointly assembled into 97,233 contigs. Among them, 23,223 contigs were mapped to known genes, of which 2,193 were predicted to be differentially expressed in ovary and 887 in testes. According to annotation information, several sex-related biological pathways including ovarian steroidogenesis and estrogen signaling pathways were firstly found in flounder. The dimorphic expression of overall sex-related genes provides further insights into sex determination and gonadal development. Our study also provides an archive for further studies of molecular mechanism of fish sex determination.