Estrodiol-17ß and aromatase inhibitor treatment induced alternations of genome-wide DNA methylation pattern in Takifugu rubripes gonads.

Estradiol-17ß (E2) and aromatase inhibitor (AI) exposure can change the phenotypic sex of fish gonads. To investigated whether alterations in DNA methylation is involved in this process, the level of genome-wide DNA methylation in Takifugu rubripes gonads was quantitatively analyzed during the E2-induced feminization and AI-induced masculinization processes in this study. The methylation levels of the total cytosine (C) in control-XX(C-XX), control-XY (C-XY), E2-treated-XY (E-XY) and AI-treated-XX (AI-XX) were 9.11%, 9.19%, 8.63% and 9.23%, respectively. In the C-XX vs C-XY comparison, 4,196 differentially methylated regions (DMRs) overlapped with the gene body of 2,497 genes and 608 DMRs overlapped with the promoter of 575 genes. In the E-XY vs C-XY comparison, 6,539 DMRs overlapped with the gene body of 3,416 genes and 856 DMRs overlapped with the promoter of 776 genes. In the AI-XX vs C-XX comparison, 2,843 DMRs overlapped with the gene body of 1,831 genes and 461 DMRs overlapped with the promoter of 421 genes. Gonadal genomic methylation mainly occurred at CG sites and the genes that overlapped with DMRs on CG context were most enriched in the signaling pathways related to gonad differentiation, such as the Wnt, TGF-ß, MAPK, CAM and GnRH pathways. The DNA methylation levels of steroid synthesis genes and estrogen receptor genes promoter or gene body were negative correlated with their expression. After bisulfite sequencing verification, the DNA methylation level of the amhr2 promoter in XY was increased after E2 treatment, which consistent with the data from the genome-wide DNA methylation sequencing. In C-XY group, the expression of amhr2 was significantly higher than that in E-XY (p < 0.05). Additionally, dnmt1, which is responsible for methylation maintenance, expressed at similar level in four groups (p > 0.05). dnmt3, tet2, and setd1b, which were responsible for methylation modification, expressed at significantly higher levels in E-XY compared to the C-XY (p < 0.05). Dnmt3 and tet2 were expressed at significantly higher levels in AI-XX than that in C-XX (p < 0.05). These results indicated that E2 and AI treatment lead to the aberrant genome-wide DNA methylation level and expression level of dnmt3, tet2, and setd1b in T. rubripes gonad.

Profile of gene expression changes during estrodiol-17ß-induced feminization in the Takifugu rubripes brain.

BACKGROUND: As the critical tissue of the central nervous system, the brain has been found to be involved in gonad development. Previous studies have suggested that gonadal fate may be affected by the brain. Identifying brain-specific molecular changes that occur during estrodiol-17ß (E2) -induced feminization is crucial to our understanding of the molecular control of sex differentiation by the brains of fish. RESULTS: In this study, the differential transcriptomic responses of the Takifugu rubripes larvae brain were compared after E2 treatment for 55 days. Our results showed that 514 genes were differentially expressed between E2-treated-XX (E-XX) and Control-XX (C-XX) T. rubripes, while 362 genes were differentially expressed between E2-treated-XY (E-XY) and Control-XY (C-XY). For example, the expression of cyp19a1b, gnrh1 and pgr was significantly up-regulated, while st, sl, tshß, prl and pit-1, which belong to the growth hormone/prolactin family, were significantly down-regulated after E2 treatment, in both sexes. The arntl1, bhlbe, nr1d2, per1b, per3, cry1, cipc and ciart genes, which are involved in the circadian rhythm, were also found to be altered. Differentially expressed genes (DEGs), which were identified between E-XX and C-XX, were significantly enriched in neuroactive ligand-receptor interaction, arachidonic acid metabolism, cytokine-cytokine receptor interaction and the calcium signaling pathway. The DEGs that were identified between E-XY and C-XY were significantly enriched in tyrosine metabolism, phenylalanine metabolism, arachidonic acid metabolism and linoleic acid metabolism. CONCLUSION: A number of genes and pathways were identified in the brain of E2-treated T. rubripes larvae by RNA-seq. It provided the opportunity for further study on the possible involvement of networks in the brain-pituitary-gonadal axis in sex differentiation in T. rubripes.

Effects of the Thyroid Endocrine System on Gonadal Sex Ratios and Sex-Related Gene Expression in the Pufferfish Takifugu rubripes.

To examine the effect and mechanism of thyroid hormone on gonadal sex differentiation, Takifugu rubripes larvae were treated with goitrogen (methimazole, MET, 1000 g/g), and thyroxine (T4, 2nM) from 25 to 80 days after hatching (dah). Gonadal histology and sex ratios of fish were then determined at 80 dah. MET treatment induced masculinization, but T4 treatment did not induce feminization in T. rubripes larvae. Transcriptomic analysis of gonads at 80 dah was then conducted. Among the large number of differentially expressed genes between the groups, the expression of foxl2, cyp19a1a, and dmrt1 was altered. The expression of foxl2, cyp19a1a, dmrt1 and gsdf at 25, 40, 55 days after treatment (dat) was further analyzed by qPCR. MET treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 in genetic females (p < 0.05). Additionally, T4 treatment induced an increase in the expression of cyp19a1a in genetic XY gonads only at 25 dat. However, the increase in cyp19a1a expression did not continue to 40 and 55 dat. This may explain why feminization of larvae was not found in the T4-treated group. Thus, the present study provides the first evidence that MET treatment causes masculinization in teleost fish. The effects of MET-induced masculinization in T. rubripes may act primarily via suppression of the expression of foxl2 and cyp19a1a, and stimulation of the expression of dmrt1. Moreover, the effects of higher concentrations of T4 or different concentrations of T3, on sex differentiation require further testing.

Identification of sex differentiation-related microRNA and long non-coding RNA in Takifugu rubripes gonads.

Although sex determination and differentiation are key developmental processes in animals, the involvement of non-coding RNA in the regulation of this process is still not clarified. The tiger pufferfish (Takifugu rubripes) is one of the most economically important marine cultured species in Asia, but analyses of miRNA and long non-coding RNA (lncRNA) at early sex differentiation stages have not been conducted yet. In our study, high-throughput sequencing technology was used to sequence transcriptome libraries from undifferentiated gonads of T. rubripes. In total, 231 (107 conserved, and 124 novel) miRNAs were obtained, while 2774 (523 conserved, and 2251 novel) lncRNAs were identified. Of these, several miRNAs and lncRNAs were predicted to be the regulators of the expression of sex-related genes (including fru-miR-15b/foxl2, novel-167, novel-318, and novel-538/dmrt1, novel-548/amh, lnc_000338, lnc_000690, lnc_000370, XLOC_021951, and XR_965485.1/gsdf). Analysis of differentially expressed miRNAs and lncRNAs showed that three mature miRNAs up-regulated and five mature miRNAs were down-regulated in male gonads compared to female gonads, while 79 lncRNAs were up-regulated and 51 were down-regulated. These findings could highlight a group of interesting miRNAs and lncRNAs for future studies and may reveal new insights into the function of miRNAs and lncRNAs in sex determination and differentiation.

Gene Expression of Takifugu rubripes Gonads During AI- or MT-induced Masculinization and E2-induced Feminization.

Elucidating the global molecular changes that occur during aromatase inhibitor (AI)- or 17α-methyltestosterone (MT)-induced masculinization and estradiol-17ß (E2)-induced feminization is critical to understanding the roles that endocrine and genetic factors play in regulating the process of sex differentiation in fish. Here, fugu larvae were treated with AI (letrozole), MT, or E2 from 25 to 80 days after hatching (dah), and gonadal transcriptomic analysis at 80 dah was performed. The expression of dmrt1, gsdf, foxl2, and other key genes (star, hsd3b1, cyp11c1, cyp19a1a, etc.) involved in the steroid hormone biosynthesis pathway were found be altered. The expression of dmrt1, gsdf, cyp19a1a, and foxl2 was further verified by quantitative polymerase chain reaction. In the control group, the expression of dmrt1 and gsdf was significantly higher in XY larvae than in XX larvae, while the expression of foxl2 and cyp19a1a was significantly higher in XX larvae than in XY larvae (P < .05). AI treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 and gsdf in XX larvae. MT treatment suppressed the expression of foxl2, cyp19a1a, dmrt1, and gsdf in XX larvae. E2 treatment suppressed the expression of dmrt1 and gsdf, but did not restore the expression of foxl2 and cyp19a1a in XY larvae. The shared response following AI, MT, and E2 treatment suggested that these genes are essential for sex differentiation. This finding offers some insight into AI or MT-induced masculinization, and E2-induced femininization in fugu.

Transcriptomic analyses reveal novel genes with sexually dimorphic expression in Takifugu rubripes brain during gonadal sex differentiation.

BACKGROUND: Quantification of mRNAs in gonads and other tissues at the early critical development stage of sex differentiation may help to provide a global view of regulatory mechanisms underlying sex differentiation. We have recently reported the transcriptomic profiling of fugu gonad associated with sex differentiation. OBJECTIVES: This study attempted to identify the genes in the brain that are involved in gonadal differentiation and development. METHODS: In this study, a transcriptomic scan of potential candidate genes involved in sex differentiation was conducted in the brains of fugu larvae at 30 and 40 dah (morphological gonadal sex differentiation had not yet occurred). The dimorphic expression patterns of several candidate genes were verified using quantitative PCR. RESULTS: A total of 28.24 Gb of clean reads were obtained and 22,337 genes were identified in the brains of fugu larvae. These included 1008 novel genes that provide abundant data for functional analysis of sex differentiation. 229 genes were identified in the 30 dah larvae that were abundant in the XY brain and 21 that were abundant in the XX brain. In the 40 dah larvae, 325 genes were identified abundant in the XY brain and 174 were identified abundant in the XX brain. CONCLUSION: This is the first investigation into the transcriptome of the fugu larvae brain at the early sex differentiation stage. The results obtained here will enhance the understanding of molecular mechanisms that underly fugu sex differentiation.

Effects of different light conditions on the retinal microstructure and ultrastructure of Dicentrarchus labrax larvae.

Light is a key environmental parameter known to influence fish throughout various stages of their life, from embryonic development to sexually mature adults. In a recent study, the effects of different light conditions on the growth of Dicentrarchus labrax larvae were investigated using light-emitting diodes (LEDs) as a light source. Here, pathological examinations were carried out to assess whether variations in light affected the visual system of the larvae, including any negative impacts on the retina or the growth rate. Although light did not affect the total thickness (TT) of the retina, the thickness of the retinal pigment epithelium layer (PRE), photoreceptor layer (PRos/is), outer nuclear layer (ONL), and inner nuclear layer (INL), and the PRE/TT and ONL/TT ratios were all significantly higher in larvae exposed to blue light than in larvae exposed to white light. Additionally, the thickness of PRE and the outer nuclear layer and the RPE/TT and ONL/TT ratios of larvae exposed to 2.0 W m-2 were significantly lower than in larvae exposed to 0.3 W m-2. By contrast, the INL/TT ratio in larvae exposed to 2.0 W m-2 was significantly higher than in larvae exposed to 0.3 W m-2. Additionally, the INL and ganglion cell layer nuclei density of larvae exposed to 2.0 W m-2 were significantly higher than in those exposed to 0.3 W m-2 (p < 0.05). Transmission electron microscopy revealed different levels of abnormalities in the photoreceptor layers in all treatment groups. Considering the growth of the larvae, the results of the study suggest that continuous LED exposure induced damage to photoreceptor cells but was not relevant to the growth performance of D. labrax larvae. Moreover, the results obtained here also support the high plasticity of retinal development in response to altered environmental light conditions.

Growth and survival of Takifugu rubripes larvae cultured under different light conditions.

We assessed the effects of light intensity and spectrum on the growth and survival of Takifugu rubripes larvae from 30 to 69 days after hatching. Five lighting regimes were applied using 0.5, 1.5, and 3.0 W m-2 full spectrum white (W0.5, W1.5, W3.0), 0.5 W m-2 yellow (Y0.5), and 0.5 W m-2 blue light (B0.5). At the end of the experiment, body length, wet weight, and specific growth rate from day 0 to day 39 were significantly greater in larvae reared under W3.0 than under B0.5 (P Ë‚ 0.05). No significant differences were observed among W0.5, W1.5, and W3.0, or among W0.5, Y0.5, and B0.5 (P > 0.05). Survival rate was significantly higher in larvae reared under W1.5 than W0.5 (P Ë‚ 0.05), but no significant differences were observed among W0.5, Y0.5, and B0.5 (P > 0.05). Additionally, light conditioning did not affect the total thickness of the retina. Although the ratio of the thickness of the retinal pigment epithelium layer/total thickness (TT) was significantly higher in larvae exposed to W3.0 compared with those exposed to other light conditions, and the thickness of the outer nuclear layer/TT was significantly lower in larvae exposed to W3.0 compared with those exposed to W0.5 (P < 0.05), no relationship was confirmed between the structure of the retina and the growth performance of the T. rubripes larvae. Expression patterns of two stress-related and seven growth-related genes were also compared with the biometric parameters investigated in the experimental groups. No significant differences in the aanat1a, crh, ss1, igf1, or igf2 expression were observed among the five treatments. Pomc expression was significantly lower in larvae exposed to W1.5 than the larvae exposed to W0.5, and it was significantly lower in larvae exposed to Y0.5 than in larvae exposed to W0.5 or B0.5 (P < 0.05). Significant differences were also found in the expression of gh, with the highest levels being observed under W3.0, while the lowest levels were observed in B0.5 (P < 0.05). Ghrh expression was significantly higher in W3.0 (P < 0.05). These results should be considered when designing rearing protocols for fugu larvae in aquaculture systems.

Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in Takifugu rubripes gonad at the early stage of sex differentiation.

Quantifying the expression of mRNAs in the gonads at the critical stage of molecular sex differentiation stage might help to clarify the regulatory network during early sex differentiation and provide new information on the role of sex-related genes in gonadal function. In this study, transcriptomic analysis of sex-related genes expression profiles in fugu gonads at 60 and 90 days after hatching (dah) was conducted firstly, and a total of 112,504,991 clean reads, encompassing 28.35 Gb of sequences were retrieved. Twenty-three thousand eight hundred ten genes were found to be expressed in juvenile fugu gonads, and we mainly focused on the differentially expressed genes that have the potential to be involved in the gonadal sex differentiation. For 60-dah juveniles, we identified 1014 genes that were upregulated in the ovary and 1570 that were upregulated in the testis. For 90-dah juveniles, we identified 1287 genes that were upregulated in the ovary and 1500 that were upregulated in the testis. The dimorphic expression patterns of 15 genes in gonads at 30 and 40 dah were further investigate using qPCR. Cyp11b and star were expressed at higher levels in XY than in XX, while cyp11a1 and cyp19a1a were expressed at higher levels in XX than in XY at 30 dah. At 40 dah, the levels of gsdf, dmrt1, dmrt3, cyp11c1, star, and hsd3b expression were higher in XY, while the levels of foxl2, cyp19a1a, wnt9b, and foxD4 expression were higher in XX. Sox9, cyp11a1, cyp17a1, cyp17a2, and nr5a2 were expressed at similar levels in XX and XY at 40 dah. This is the first report of gonadal transcriptome of fugu at early sex differentiation stage, and our results provide an archive for further study on molecular mechanism underlying sex differentiation in this species.

De novo transcriptome analysis and differentially expressed genes in the ovary and testis of the Japanese mantis shrimp Oratosquilla oratoria by RNA-Seq.

The mantis shrimp Oratosquilla oratoria is a widely distributed, commercially important crustacean species. Although its conservation and the development of successful artificial breeding technologies have recently received considerable attention, there are currently no available data regarding the molecular mechanisms in controlling reproduction. In this study, we performed transcriptome sequencing of the testis, ovary, female and male eyestalks and the androgenic gland of O. oratoria, and compared the expression pattern of transcripts from the testis and ovary libraries to identify genes involved in gonadal development. A total of 147,130,937 clean reads were retrieved after removing the adapters in reads and filtering out low-quality data. All the reads were assembled into 94,990 unigenes (23,133 in testis and ovary) with an average length of 783 base pairs (bp) and N50 of 1502 bp. A search of all-unigenes against COG, GO, KEGG, KOG, Pfam, Swiss-Prot and Nr databases resulted in a total of 19,404 annotated unigenes. Comparison of the sequences in the ovary and testis libraries revealed that 1188 unigenes were up-regulated in the ovary and 2732 were up-regulated in the testis. Twenty ovary-up-regulated and 21 testis-up-regulated unigenes were confirmed by quantitative real-time PCR. Additionally, 13,437 simple sequence repeats (SSRs) and 275,799 putative single nucleotide polymorphisms (SNPs) were identified. The important functional genes and pathways identified here provide a valuable dataset for understanding the molecular mechanisms controlling gonad development in O. oratoria, and the numerous (13,437 SSRs and 275,799 SNPs) molecular markers obtained here will provide fundamental basis for functional genomic and population genetic studies of O. oratoria.