Mettl3 Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish

Abstract

N⁶-methyladenosine (m⁶A), catalyzed by Mettl3 methyltransferase, is a highly conserved epigenetic modification in eukaryotic messenger RNA (mRNA). Previous studies have implicated m⁶A modification in multiple biological processes, but the in vivo function of m⁶A has been difficult to study, because mettl3 mutants are embryonic lethal in both mammals and plants. In this study, we have used transcription activator-like effector nucleases and generated viable zygotic mettl3 mutant, Zmettl3^m/m , in zebrafish. We find that the oocytes in Zmettl3^m/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. Human chorionic gonadotropin-induced ovarian germinal vesicle breakdown in vitro and the numbers of eggs ovulated in vivo are both decreased as well, while the defects of oocyte maturation can be rescued by sex hormone in vitro and in vivo In Zmettl3^m/m adult males, we find defects in sperm maturation and sperm motility is significantly reduced. Further study shows that 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels are significantly decreased in Zmettl3^m/m , and 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 Zmettl3^m/m Thus, our study provides the first in vivo evidence that loss of Mettl3 leads to failed gamete maturation and significantly reduced fertility in zebrafish. Mettl3 and m⁶A modifications are essential for optimal reproduction in vertebrates.

Keywords: N6-methyladenosine; fertility; gamete maturation; mettl3; zebrafish.

Figure 1

The expression pattern of mettl3 and m⁶A level in zebrafish embryos and adult tissues. (A and B) qPCR analysis for the temporal and spatial expression profile of mettl3 mRNA in WT embryos (A) and adult tissues (4 months postfertilization) (B). (C and D) Quantification of the m⁶A/A ratio of the total mRNA purified from WT embryos (C) and adult tissues (D) by LC-MS/MS. hpf, hours postfertilization.

Figure 2

Localization of mettl3 expression in the ovary and testis by fluorescent in situ hybridization. Cell nuclei from somatic cells are indicated by arrowheads, Leydig cells are indicated by asterisks, and Sertoli cells are indicated by arrows. NC, negative control using sense probe.

Figure 3

Establishment of mettl3 knockout mutant lines. (A) Schematic representation of the genomic structures of zebrafish mettl3 and the target sites of TALENs. Recognition sequences are boxed, and the spacer sequences are between the two black boxes. The start codon of translation ATG is underlined. Deletions and insertions are indicated by dotted line and red letters, respectively. (B) Western blot analysis of whole zebrafish and dissected testes from WT and mettl3 mutant adults. (C) Quantification of m⁶A/A ratio of the total mRNA purified from WT and mettl3 mutant adult tissues by LC-MS/MS. (Student’s t-test, *** P < 0.001; data are presented as mean ± SEM.)

Figure 4

Oocyte maturation defects in Zmettl3^m/m females. (A) Appearance of ovaries dissected from WT and Zmettl3^m/m females. (B) Gross anatomical appearance of ovaries from WT and Zmettl3^m/m females. Bar, 5 mm. (C) The GSI scatterplot of WT and Zmettl3^m/m females (n = 12). GSI, gonadosomatic index. (D and E) H&E staining of the ovaries from WT and Zmettl3^m/m females. PG follicles are indicated by arrowheads, and PV follicles are indicated by arrows. PG, primary growth stage; PV, previtellogenic stage; EV, early vitellogenic stage; MV, midvitellogenic stage; FG, full-grown stage. Bar, 200 μm. (F) The relative distribution of different stage follicles in the WT and Zmettl3^m/m females (n = 12) (* P < 0.05; data are presented as mean ± SEM).

Figure 5

Defects of oocyte maturation in mutants can be rescued by sex hormone. (A) Morphology of FG follicles dissected from WT and Zmettl3^m/m ovaries with incubation of hCG (100 IU/ml) after 6 hr. Follicles undergoing GVBD are marked by red arrows. Bar, 500 μm. (B) Comparison of the %GVBD in WT and Zmettl3^m/m in control or hCG treatment. (C) Gross morphology of ovaries dissected from adult zebrafish 4 hr after injection of isotonic saline (control) or 17α-20β-DHP. Representative follicles undergoing GVBD are marked by red arrows. Bar, 500 μm. (D) Quantitative assessment of oocyte maturation induction by injection of 17α-20β-DHP. (n = 12, one-way ANOVA; data are presented as mean ± SEM.)

Figure 6

Sperm maturation is affected in Zmettl3^m/m male. (A) Appearance of ovaries dissected from WT and Zmettl3^m/m males. (B) Gross morphological appearance of ovaries from WT and Zmettl3^m/m males. Bar, 5 mm. (C) The GSI scatterplot of WT and Zmettl3^m/m males. (D and E) H&E staining of testes from WT and Zmettl3^m/m. SG, spermatogonia; SC, spermatocyte; ST, spermatid; SZ, spermatozoa. SG are marked by arrows. Bar, 100 μm. (F) The ratio of different stage sperms in the WT and Zmettl3^m/m males. (n = 15, Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001; data are presented as mean ± SEM.)

Figure 7

Synapsis proceeds normally in the Zmettl3^m/m spermatocytes. Bar, 10 μm (n = 12).

Figure 8

Fertility and sex ratios are altered in Zmettl3^m/m adults. (A) The fertilization rate (fertilization rate = fertilized eggs/total eggs × 100%) of WT, Zmettl3^m/m, and OE-KO adults. ♀, female, ♂, male (n = 30, one-way ANOVA). (B) The ovulation rate (ovulation rate = number of spawned females/total number of females × 100%) of WT, Zmettl3^m/m, and OE-KO females crossed with WT males (n = 30, Student’s t-test). (C) The numbers of eggs ovulated by WT, Zmettl3^m/m, and OE-KO females crossed with WT males (n = 30, Student’s t-test). (D) The ovulation rate of WT females crossed with WT, Zmettl3^m/m, and OE-KO males (n = 30, Student’s t-test). (E) Sex ratio of WT, Zmettl3^m/m, and OE-KO adults (n = 120, one-way ANOVA; data are presented as mean ± SEM).

Figure 9

Gene expression profiles in WT and Zmettl3^m/m adults. Expression of genes in pituitaries of females (A) and males (C), FG stage follicles (B), and testes (D) by qPCR. (Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001; data are presented as mean ± SEM.)

Figure 10

Serum 11-KT and E2 level are significantly decreased in Zmettl3^m/m adults. (A) Serum concentration of 11-KT and E2 in WT and Zmettl3^m/m females. (B) Serum concentration of 11-KT and E2 in WT and Zmettl3^m/m males. (n = 12, Student’s t-test, * P < 0.05, *** P < 0.001; data are presented as mean ± SEM.)