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Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes.

Tao W, Yuan J, Zhou L, Sun L, Sun Y, Yang S, Li M, Zeng S, Huang B, Wang D - PLoS ONE (2013)

Bottom Line: Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads.Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads.Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, PR China.

ABSTRACT
Four pairs of XX and XY gonads from Nile tilapia were sequenced at four developmental stages, 5, 30, 90, and 180 days after hatching (dah) using Illumina Hiseq(TM) technology. This produced 28 Gb sequences, which were mapped to 21,334 genes. Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads. Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads. Almost all steroidogenic enzymes, including cyp19a1a, were up-regulated in XX gonads at 5 dah; but in XY gonads these enzymes, including cyp11b2, were significantly up-regulated at 90 dah, indicating that, at a time critical to sex determination, the XX fish produced estrogen and the XY fish did not produce androgens. The most pronounced expression of steroidogenic enzyme genes was observed at 30 and 90 dah for XX and XY gonads, corresponding to the initiation of germ cell meiosis in the female and male gonads, respectively. Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah. This could explain why exogenous steroid treatment induced XX and XY sex reversal. The XX-enhanced expression of cyp19a1a and cyp19a1b at all stages suggests an important role for estrogen in female sex determination and maintenance of phenotypic sex. This work is the largest collection of gonadal transcriptome data in tilapia and lays the foundation for future studies into the molecular mechanisms of sex determination and maintenance of phenotypic sex in non-model teleosts.

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Pipeline of experiment (A) and bioinformatics analysis (B).
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pone-0063604-g001: Pipeline of experiment (A) and bioinformatics analysis (B).

Mentions: Total RNA was extracted from each sample using Trizol Reagent (Invitrogen, Carlsbad, CA, U.S.) according to the manufacturer's instructions. The extracted RNA was further treated with DNase1 (RNase-free 5 U/μL) to eliminate genomic DNA contamination. The concentration and integrity of RNA were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, U.S.) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, U.S.). The RNA with OD260/280 = 1.8–2.1, OD260/230≥1.7, c≥150 ng/µL, RNA integrity number (RIN)≥7.0 and 28S/18S>1.0 were performed for mRNA enrichment using oligo (dT) beads. The enriched mRNA was disrupted into short fragments (200–700 nt) using fragmentation buffer. These short fragments were used as templates, first-strand cDNA was synthesized using an Invitrogen cDNA synthesis kit (Invitrogen, Carlsbad, CA, U.S.), followed by synthesis of second-strand cDNA using custom second-strand synthesis buffer, dNTPs, RNase H, and DNA polymerase I. A QiaQuick PCR purification kit (Qiagen GmbH, Hilden, Germany) was used to purify these fragments and EB buffer was used for end repair and addition of the poly (A) tail. Then these short fragments were ligated with sequencing adapters. After agarose gel electrophoresis, fragments between 320 and 370 nt were cut from the gel for PCR amplification. In total, eight cDNA libraries were constructed from the eight respective samples. Flow chart A in Figure 1 shows the major steps of the experiment.


Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes.

Tao W, Yuan J, Zhou L, Sun L, Sun Y, Yang S, Li M, Zeng S, Huang B, Wang D - PLoS ONE (2013)

Pipeline of experiment (A) and bioinformatics analysis (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643912&req=5

pone-0063604-g001: Pipeline of experiment (A) and bioinformatics analysis (B).
Mentions: Total RNA was extracted from each sample using Trizol Reagent (Invitrogen, Carlsbad, CA, U.S.) according to the manufacturer's instructions. The extracted RNA was further treated with DNase1 (RNase-free 5 U/μL) to eliminate genomic DNA contamination. The concentration and integrity of RNA were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, U.S.) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, U.S.). The RNA with OD260/280 = 1.8–2.1, OD260/230≥1.7, c≥150 ng/µL, RNA integrity number (RIN)≥7.0 and 28S/18S>1.0 were performed for mRNA enrichment using oligo (dT) beads. The enriched mRNA was disrupted into short fragments (200–700 nt) using fragmentation buffer. These short fragments were used as templates, first-strand cDNA was synthesized using an Invitrogen cDNA synthesis kit (Invitrogen, Carlsbad, CA, U.S.), followed by synthesis of second-strand cDNA using custom second-strand synthesis buffer, dNTPs, RNase H, and DNA polymerase I. A QiaQuick PCR purification kit (Qiagen GmbH, Hilden, Germany) was used to purify these fragments and EB buffer was used for end repair and addition of the poly (A) tail. Then these short fragments were ligated with sequencing adapters. After agarose gel electrophoresis, fragments between 320 and 370 nt were cut from the gel for PCR amplification. In total, eight cDNA libraries were constructed from the eight respective samples. Flow chart A in Figure 1 shows the major steps of the experiment.

Bottom Line: Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads.Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads.Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, PR China.

ABSTRACT
Four pairs of XX and XY gonads from Nile tilapia were sequenced at four developmental stages, 5, 30, 90, and 180 days after hatching (dah) using Illumina Hiseq(TM) technology. This produced 28 Gb sequences, which were mapped to 21,334 genes. Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads. Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads. Almost all steroidogenic enzymes, including cyp19a1a, were up-regulated in XX gonads at 5 dah; but in XY gonads these enzymes, including cyp11b2, were significantly up-regulated at 90 dah, indicating that, at a time critical to sex determination, the XX fish produced estrogen and the XY fish did not produce androgens. The most pronounced expression of steroidogenic enzyme genes was observed at 30 and 90 dah for XX and XY gonads, corresponding to the initiation of germ cell meiosis in the female and male gonads, respectively. Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah. This could explain why exogenous steroid treatment induced XX and XY sex reversal. The XX-enhanced expression of cyp19a1a and cyp19a1b at all stages suggests an important role for estrogen in female sex determination and maintenance of phenotypic sex. This work is the largest collection of gonadal transcriptome data in tilapia and lays the foundation for future studies into the molecular mechanisms of sex determination and maintenance of phenotypic sex in non-model teleosts.

Show MeSH