Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish

Klangnurak, Wanlada; Fukuyo, Taketo; Rezanujjaman, Md.; Seki, Masahide; Sugano, Sumio; Suzuki, Yutaka; Tokumoto, Toshinobu

doi:10.1371/journal.pone.0196544

Cited by 8 publications

(4 citation statements)

References 42 publications

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“…Levels of certain proteins and lipids have been linked to egg viability in many fish species including rainbow trout [16]. A relationship between the maternal transcriptome and developmental competence has also been supported in a variety of fishes using an assortment of molecular approaches, although few investigations involved next-generation sequencing [17, 18]. In rainbow trout, genes linked to decreased egg quality caused by postovulatory aging were identified using quantitative reverse transcription PCR [19–21] and transcripts associated with decreased egg quality in response to the use of photoperiod to shorten the time to spawning or hormone-induced ovulation were identified by microarray analyses [22, 23].…”

Section: Introductionmentioning

confidence: 99%

Transcriptome analysis of egg viability in rainbow trout, Oncorhynchus mykiss

Martin

Dixon

et al. 2019

BMC Genomics

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Background Maternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries. Results The percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30–50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses. Conclusions Our results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs. Electronic supplementary material The online version of this article (10.1186/s12864-019-5690-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Transcriptome analysis of egg viability in rainbow trout, Oncorhynchus mykiss

Martin

Dixon

et al. 2019

BMC Genomics

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“…In addition to the morphology of otoliths, we had already found that eggs from stm -/mutants showed a high nonfertilization rate (Klangnurak et al, 2018). By paring stm -/mutants in later generations (F3 to F5), we obtained high percentages of unfertilized eggs.…”

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confidence: 71%

“…Although the fertilization rate decreased, ovulation could be induced in our stm homozygous mutant fish, which demonstrated that stm was not an ovulation-inducing gene (Klangnurak et al, 2018). In this study, further analysis of the stm mutant strain was conducted.…”

Section: Introductionmentioning

confidence: 88%

“…By using an in vivo induction method for oocyte maturation and ovulation in zebrafish by adding compounds to water (Tokumoto et al, 2011), we identified 11 genes that were highly upregulated during the induction of ovulation (Klangnurak et al, 2018). Then, we determined the functions of these genes by establishing gene knockout zebrafish strains of these genes.…”

Section: Introductionmentioning

confidence: 99%

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Zebrafish stm is involved in the development of otoliths and of the fertilization envelope

Pachoensuk

Fukuyo

Rezanujjaman

et al. 2021

Reproduction and Fertility

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Using an in vivo assay, we selected 11 genes that were highly upregulated during the induction of ovulation in zebrafish using microarray analysis and RNA sequencing. The Starmaker gene (stm) was one of these genes. Although stm has been previously reported to be involved in otolith formation during the early development of zebrafish, we detected its expression in eggs and showed that stm was related to fertilization by establishing an stm gene knockout strain using the CRISPR/Cas9 system. Further phenotypic analysis of stm knockout fish was conducted in this study. With a higher nonfertilization rate, the stm mutant strain showed an extremely low survival rate. Otoliths of stm homozygous mutant zebrafish showed abnormal morphology in embryos and adult fish. However, fish did not show any abnormalities in swimming behaviour in either embryos or adults. Stm proteins were detected on the chorion of ovulated eggs before spawning. Fibre-supported knob-like structures on the fertilization envelope (FE) also showed abnormal structures in stm mutants. The Stm protein is necessary for otolith formation, and a lack of Stm causes abnormal otolith formation. The partial defect of otolith formation does not cause defects in swimming behaviour. The Stm protein is expressed in the chorion and is responsible for the formation of fibre-supported knob-like structures on the FE. It was suggested that a lack of Stm caused a lower fertilization rate due to inadequate formation of the FE.

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