Pax2a is expressed in oocytes and is responsible for early development and oogenesis in zebrafish.

Eleven genes, including pax2a, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. The purpose of this study was to investigate the role of the pax2a gene in the ovulation-inducing process. F2 pax2a homozygous mutant zebrafish possessing a deletion of 6 nucleotides were established in this study. However, the deletion included the start codon (ATG) of the pax2a gene, and the Pax2a protein was still detected, which indicated that the deletion caused a shift in the start codon to the next ATG, resulting in a 12-amino acid deletion. F2 pax2a homozygous mutant zebrafish showed ovulation. However, the embryos showed an abnormal oval shape at the epiboly stage that resulted in yolk and tail formation abnormalities and heart edema. The surviving F3 homozygous mutants did not develop ovaries. Pax2a was detected in oocytes and eggs but not after the Prim-22 stage. It is suggested that pax2a is expressed as a maternal gene in oocytes and is necessary for oogenesis and early development.

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

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.

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

We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm), were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.