Abstract
SummaryUsing 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.Lay summaryIn zebrafish, the protein Starmaker (Stm) was identified as having a role in ovulation. Stm is also known to be required for the formation of ear stones (otoliths) which are needed to keep the body in balance. Zebrafish lacking Stm were produced by genome editing. As expected, Stm-deficient fish formed abnormal otoliths. To investigate the role of Stm in ovulation, fertilization and early development, we tried mating of Stm mutants and observed their juveniles. Although no problem found in ovulation, we found low fertilization rate and abnormal structure of knob-like structure (small pit) on the egg membrane. Survival rate of embryos with abnormal egg membrane was extremely low. It was demonstrated that Stm protein is necessary to form the functional egg membrane to protect embryos from the outside environment.
Highlights
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)
Three types of otoliths, pebbles (Lapillus), flat stones (Sagitta), and stellates (Asteriscus), developed in the stm−/− mutant and the WT fish. The shape of these otoliths was abnormal in all the stm−/− mutants fishes examined (Fig. 3)
Relatively similar shapes were observed in the case of Sagitta and Asteriscus, the surfaces of these stones were uneven in the stm−/− mutant compared with the wild type
Summary
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). We have already established genome-modified fish for the starmaker (stm) gene. The Starmaker (stm) gene has been reported to be responsible for the formation of otoliths in zebrafish (Sollner et al 2003). Otoliths contain calcium carbonate and some matrix proteins, among which Stm is known to play an important role in changing the crystal structure of calcium carbonate from calcite to aragonite (Sollner et al 2003). It is known that there are three types of crystal structures of calcium carbonate, calcite, aragonite, and vaterite, and calcite is the most stable crystal state at normal temperature and pressure. It is believed that this causes strong binding to calcium ions, which in turn causes a change in crystal structure from calcite to aragonite (Kalka et al 2019)
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