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

Theeranukul Pachoensuk; Taketo Fukuyo; Md Rezanujjaman; Klangnurak Wanlada; Chihiro Yamamoto; Akiteru Maeno; Md Mostafizur Rahaman; Md Hasan Ali; Toshinobu Tokumoto

doi:10.1530/RAF-20-0040

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

Reprod Fertil. 2021 Feb 16;2(1):7-16. doi: 10.1530/RAF-20-0040. eCollection 2021 Jan.

Authors

Affiliations

¹ Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation, Shizuoka University, Suruga-ku, Shizuoka, Japan.
² Biological Science Course, Graduate School of Science, Shizuoka University, Suruga-ku, Shizuoka, Japan.
³ Department of Animal Production and Fisheries, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand.
⁴ Division of Technical Service, Shizuoka University, Suruga-ku, Shizuoka, Japan.
⁵ Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka, Japan.
⁶ Facility and Equipment Technical Unit, National Institute of Genetics, Mishima, Shizuoka, Japan.

Abstract

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.

Lay summary: In 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.

Keywords: fertilization; fertilization envelope; otoliths; starmaker; zebrafish.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Female
Fertilization
Gene Knockout Techniques
Otolithic Membrane*
Zebrafish Proteins
Zebrafish*

Substances

Zebrafish Proteins