Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.

Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus).

As an important maricultured fish, the olive flounder Paralichthys olivaceus shows sex-dimorphic growth. Thus, the molecular mechanisms involved in sex control in P. olivaceus have attracted researchers' attention. Among the sex-related genes, forkhead box protein L2 (foxl2) exhibits significant sex-dimorphic expression patterns and plays an important role in fish gonad differentiation and development. The present study first investigated the expression levels and promoter methylation dynamics of foxl2 during flounder gonad differentiation under treatments of high temperature and exogenous 17ß-oestradiol (E2). During high temperature treatment, the expression of flounder foxl2 may be repressed via maintenance of DNA methylation. Then, flounder with differentiated testis at Stages I-II were treated with exogenous 5ppm E2 or 5ppm E2+150ppm trilostane (TR) to investigate whether exogenous sex hormones could induce flounder sex reversal. The differentiated testis exhibited phenotypic variations of gonadal dysgenesis with upregulation of female-related genes (foxl2 and cytochrome P450 family 19 subfamily A (cyp19a)) and downregulation of male-related genes (cytochrome P450 family 11 subfamily B member 2 (cyp11b2), doublesex- and mab-3 related transcription factor 1 (dmrt1), anti-Mullerian hormone (amh) and SRY-box transcription factor 9 (sox9)). Furthermore, a cotransfection assay of the cells of the flounder Sertoli cell line indicated that Foxl2 was able alone or with nuclear receptor subfamily 5 group A member 2 (Nr5a2) jointly to upregulate expression of cyp19a. Moreover, Foxl2 and Nr5a2 repressed the expression of dmrt1. In summary, Foxl2 may play an important role in ovarian differentiation by maintaining cyp19a expression and antagonising the expression of dmrt1. However, upregulation of foxl2 is not sufficient to induce the sex reversal of differentiated testis.

Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus.

The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17ß-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 µM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 µM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.

Significant association of cyp19a promoter methylation with environmental factors and gonadal differentiation in olive flounder Paralichthys olivaceus.

The sex ratio of olive flounder Paralichthys olivaceus is sensitive to temperature or exogenous hormone exposures in the period of gonadal differentiation. Among sex-related genes, cyp19a, encoding cytochrome P450 aromatase, exhibits significant sex-dimorphic expression pattern and plays an important role in fish gonadal differentiation and development. The present study investigated the expression levels and promoter methylation dynamics of cyp19a and its regulators (nr5a2 and nr0b1), and sex-steroid hormone levels during flounder gonadal differentiation under the treatments of high temperature and estradiol-17ß (E2). The results showed that levels of flounder cyp19a expression and estradiol-17ß were repressed by high temperature treatment during this period. The up-regulation of nr5a2 by E2 treatment may be related to the all-female formation, and up-regulation of nr0b1 by high temperature treatment may be associated with masculinization. Co-transfection assay indicated that nr5a2 and nr0b1 were antagonist regulators of cyp19a. Furthermore, cyp19a promoter exhibited significant demethylation phenomenon at early stage of ovarian differentiation. While, high temperature could repress the demethylation process, resulting in hypermethylation maintenance in cyp19a promoter. The hypermethylation promoter was able to suppress cyp19a expression by blocking the nr5a2-mediated transactivation activity in vitro. The DNA methylation of epigenetic modification in cyp19a promoter might be the vital way linking environmental factors and gonadal differentiation in flounder.

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.

Tree characteristics and drought severity modulate the growth resilience of natural Mongolian pine to extreme drought episodes.

Global climate change and the increase in the frequency and intensity of drought have led to widespread forest decline and tree mortality. Studying the resilience components of tree growth to drought, including resistance (Rt), recovery (Rc), and resilience (Rs) and the influencing factors, helps assess forests' production and ecological stability under a changing climate. This study analyzed the responses of three resilience components of natural Mongolian pine (Pinus sylvestris var. mongolica) to drought events by examining individual-tree characteristics in two sites of Hulunbuir using the linear mixed effect model. The result showed that drought severity, diameter at breast height (dbh), pre-drought growth, and growth variability prior to drought had significant effects on the three resilience components of Mongolian pine growth. Specifically, as drought severity, dbh and growth variability increased, the Rt and Rs decreased, but Rc increased, showing a trade-off relationship with Rt. However, the Rt, Rc, and Rs decreased with pre-drought growth. Inter-tree competition and tree age also significantly impacted two resilience components. Besides, the interaction term between tree competition and tree age negatively affects Rt and Rs but positively affects Rc. Our findings highlight the influence of drought severity and individual-tree characteristics on drought resilience components, which can serve the adaptive management of natural Mongolian pine forests in the future.

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.

Characterization of Embryo Transcriptome of Gynogenetic Olive Flounder Paralichthys olivaceus.

Olive flounder Paralichthys olivaceus is an important commercially cultured marine flatfish in China, Korea, and Japan. Gynogenesis, via meiogynogenesis and mitogynogenesis, shows advantages in breeding and sex control, but the low survival rate, especially for mitogynogenesis, limits its application. In this study, we sequenced the embryo transcriptomes of gynogenetic haploid, meiogynogenetic diploid, mitogynogenetic diploid, and common diploid flounder and investigated their respective genetic characteristics by analyzing differentiated expressed genes. Compared with common diploid, the gynogenetic haploid showed significant downregulation in notch signaling and wingless-related integration site (Wnt) signaling pathways, which may be the source of haploid syndrome. In both meiogynogenesis and mitogynogenesis, several upregulated genes including complement C3, formin-2, and intelectin may be related to increased survival compared to the haploid. The downregulation of immune system and energy metabolism-related genes caused retarded development of gynogenetic diploids compared with the common diploid. These data provided new and important information for application of artificially induced gynogenesis to aquaculture.

Classification and Identification of Plant Fibrous Material with Different Species Using near Infrared Technique-A New Way to Approach Determining Biomass Properties Accurately within Different Species.

Plant fibrous material is a good resource in textile and other industries. Normally, several kinds of plant fibrous materials used in one process are needed to be identified and characterized in advance. It is easy to identify them when they are in raw condition. However, most of the materials are semi products which are ground, rotted or pre-hydrolyzed. To classify these samples which include different species with high accuracy is a big challenge. In this research, both qualitative and quantitative analysis methods were chosen to classify six different species of samples, including softwood, hardwood, bast, and aquatic plant. Soft Independent Modeling of Class Analogy (SIMCA) and partial least squares (PLS) were used. The algorithm to classify different species of samples using PLS was created independently in this research. Results found that the six species can be successfully classified using SIMCA and PLS methods, and these two methods show similar results. The identification rates of kenaf, ramie and pine are 100%, and the identification rates of lotus, eucalyptus and tallow are higher than 94%. It is also found that spectra loadings can help pick up best wavenumber ranges for constructing the NIR model. Inter material distance can show how close between two species. Scores graph is helpful to choose the principal components numbers during the model construction.

The testis and ovary transcriptomes of the rock bream (Oplegnathus fasciatus): A bony fish with a unique neo Y chromosome.

The rock bream (Oplegnathus fasciatus) is considerably one of the most economically important marine fish in East Asia and has a unique neo-Y chromosome system that is a good model to study the sex determination and differentiation in fish. In the present study, we used Illumina sequencing technology (HiSeq2000) to sequence, assemble and annotate the transcriptome of the testis and ovary tissues of rock bream. A total of 40,004,378 (NCBI SRA database SRX1406649) and 53,108,992 (NCBI SRA database SRX1406648) high quality reads were obtained from testis and ovary RNA sequencing, respectively, and 60,421 contigs (with average length of 1301 bp) were obtained after de novo assembling with Trinity software. Digital gene expression analysis reveals 14,036 contigs that show gender-enriched expressional profile with either testis-enriched (237 contigs) or ovary-enriched (581 contigs) with RPKM > 100. There are 237 male- and 582 female-abundant expressed genes that show sex dimorphic expression. We hope that the gonad transcriptome and those gender-enriched transcripts of rock bream can provide some insight into the understanding of genome-wide transcriptome profile of teleost gonad tissue and give useful information in fish gonad development.

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.