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Gamete Maturation in Zebrafish Requires m6A Modification Catalyzed by Mettl3 Methyltransferase
N6-methyladenosine (m6A), catalyzed by methyltransferase-like 3(Mettl3), is the most abundant RNA modification and has implicated in a variety of biological processes. However, much less is known about its function in vivo because the mutation of mettl3 are embryonic lethal in both mammals and plants. The Research Group of Fish Gene Engineering at Institute of Hydrobiology (IHB) of Chinese Academy of Sciences has obtained the mettl3 knockout zebrafish and found that mettl3 mutation disrupted gamete maturation. This study provides the first in vivo evidence that gamete maturation in zebrafish requires m6A modification catalyzed by Mettl3 methyltransferase.
In the study, IHB researchers obtained viable zygotic mettl3 knockout zebrafish line Zmettl3m/m by transcription activator-like effector nucleases (TALENs) and generated Mettl3 overexpressing transgenic zebrafish lines to rescue the homozygotes. They analyzed the various process during gametogenesis and gamete maturation in Zmettl3m/m, such as histological analysis and fertility assessment, ovarian follicles isolation and incubation, sperm motility assessments and sex steroid measurements.
These researchers find the oocytes in Zmettl3m/m adult females are stalled in early development and the ratio of full grown stage (FG) follicles is significantly lower than that of wild type (WT). In addition, the percentage of germinal vesicle breakdown (GVBD) and numbers of eggs ovulated in mutant are also significantly lower than WT follicles. While the disruption of gamete maturation was successfully rescued by overexpression of Mettl3 in Zmettl3m/m.
They also find the synaptonemal complex can be assembled and synapsis is normal in Zmettl3m/m adult males. However, the proportion of spermatogonia (SG) and spermatocytes (SC) stages are significantly higher, yet the proportion of spermatozoa (SZ) stage decrease. In addition, sperm motility is significantly reduced in mutant males. Sex steroid measurements show that 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels are significantly decreased in Zmettl3m/m. Further study shows that the defective gamete maturation is accompanied by decreased overall m6A modification levels and disrupted expression of genes critical for sex hormone synthesis and gonadotropin signaling in Zmettl3m/m.
In contrast to previous studies in mammals and plants, this study provides functional in vivo support for the theory that m6A is essential for zebrafish reproduction, highlighting a key role for m6A modification in gamete maturation.
This work is now available online in the journal GENETICS with the title of "Mettl3 mutation disrupts gamete maturation and reduces fertility in zebrafish ".
The paraffin sections from zebrafish gonad (left) and the m6A level in adult tissues (left).
A. Hematoxylin eosin (HE) staining of the ovaries from WT and Zmettl3m/m females. B. HE staining of testes from WT and Zmettl3m/m males. C. Quantification of m6A/A ratio of the total mRNA purified from WT and Zmettl3m/m adult tissues by LC-MS/MS. (Image by IHB)
