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. 2022 Jan 25:12:819856.
doi: 10.3389/fphys.2021.819856. eCollection 2021.

Glufosinate-Ammonium Induced Aberrant Histone Modifications in Mouse Sperm Are Concordant With Transcriptome in Preimplantation Embryos

Xuan Ma  1   2 , Yun Fan  1   2   3 , Wenwen Xiao  1   2 , Xingwang Ding  1   2 , Weiyue Hu  1   4 , Yankai Xia  1   2
Affiliations

Glufosinate-Ammonium Induced Aberrant Histone Modifications in Mouse Sperm Are Concordant With Transcriptome in Preimplantation Embryos

Xuan Ma et al. Front Physiol. .

Abstract

Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its male reproductive toxicity. Abnormalities in sperm histone modification induced by GLA exposure observed in our previous study aroused our interest in whether such alterations could further affect embryonic gene expression. Here we administered adult male mice with 0.2 mg/kg⋅day of GLA for 5 weeks to collect their sperm or 4-cell embryos after copulation. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing showed alterations of sperm H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), which are active histone modification marks involved in embryo development, while RNA sequencing identified differentially expressed genes in 4-cell embryos. Differentially H3K4me3 and H3K27ac occupied regions were mainly distributed at the gene promoters and putative enhancers, and were enriched in pathways related to the immune system and nervous system. Integrative analysis of these sequencing data showed that genes such as Mgl2 with increased H3K4me3 and H3K27ac in sperm were up-regulated in embryos, and vice versa for genes such as Dcn. Additionally, differentially occupied H3K4me3 and H3K27ac in sperm were linked to gene expression changes in both paternal and maternal alleles of 4-cell embryos. In conclusion, GLA-induced changes in sperm H3K4me3 and H3K27ac are concordant with gene expression in preimplantation embryos, which might further affect embryo development and offspring health.

Keywords: H3K27ac; H3K4me3; embryo development; glufosinate-ammonium; sperm.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cluster characteristics of integrated H3K4me3 and H3K27ac in CON sperm. (A) Heatmap (left) of k-means clustering of genome-wide H3K4me3 and H3K27ac signals ± 2.0 kb the center of H3K4me3 and H3K27ac regions in CON sperm, and bar graph (right) showing distribution of regions in Cluster 1–3 relative to the nearest transcription start site (TSS). For each cluster, genes were arranged in order of decreasing signals from top to bottom. (B–D) Selected significant pathways from Gene Ontology analysis on genes from Cluster 1 (B), Cluster 2 (C) and Cluster 3 (D) in CON sperm. (E–G) Integrative Genomics Viewer tracks showing H3K4me3 and H3K27ac signals on Jun (E) from Cluster 1, Cish (F) from Cluster 2 and Phlpp1 (G) from Cluster 3 in CON and GLA sperm.
FIGURE 2
FIGURE 2
GLA-induced H3K4me3 alterations in mouse sperm. (A) Correlation matrix showing Pearson correlation coefficient of H3K4me3 between replicates of CON and GLA sperm. (B) Selection of regions with P-value < 0.05. The black dashed line corresponds to the boundary of P-value = 0.05. (C) Violin plots showing increased and decreased H3K4me3 in GLA sperm. Two replicates in each group are in the same color. (D,E) Pie chart showing the genomic distribution of regions with increased (D) and decreased (E) H3K4me3 signals in GLA sperm. (F,G) Selected significant pathways from Gene Ontology analysis on genes with increased (F) or decreased (G) H3K4me3 signals in GLA sperm. (H) Integrative Genomics Viewer tracks showing increased H3K4me3 on Fasn in GLA sperm. (I) Integrative Genomics Viewer tracks showing decreased H3K4me3 on Chd9 in GLA sperm. Pink box indicates regions with differential H3K4me3 signals (P-value < 0.05).
FIGURE 3
FIGURE 3
GLA-induced H3K27ac alterations in mouse sperm. (A) Correlation matrix showing Pearson correlation coefficient of H3K27ac between replicates of CON and GLA sperm. (B) Selection of regions with P-value < 0.05. The black dashed line corresponds to the boundary of P-value = 0.05. (C) Violin plots showing increased and decreased H3K27ac in GLA sperm. Two replicates in each group are in the same color. (D,E) Pie chart showing the genomic distribution of regions with increased (D) and decreased (E) H3K27ac signals in GLA sperm. (F,G) Selected significant pathways from Gene Ontology analysis on genes with increased (F) or decreased (G) H3K27ac signals in GLA sperm. (H) Integrative Genomics Viewer tracks showing increased H3K27ac on Gata4 in GLA sperm. (I) Integrative Genomics Viewer tracks showing decreased H3K27ac on Gyg in GLA sperm. Blue box indicates regions with differential H3K27ac signals (P-value < 0.05).
FIGURE 4
FIGURE 4
Differential sperm mRNA transcripts in connection with H3K4me3 and H3K27ac alterations in GLA sperm. (A) Heatmap of normalized mRNA transcripts in CON and GLA sperm (n = 10, data from Ma et al., 2021). (B,C) Selected significant pathways from Gene Ontology analysis on up-regulated (B) or down-regulated (C) genes in GLA sperm. (D) Venn diagram showing the number of regions with different mRNA transcripts levels in GLA sperm that overlap regions with altered H3K4me3 and H3K27ac levels. Left, up-regulated regions overlapping regions with increased H3K4me3 and H3K27ac. Right, down-regulated regions overlapping regions with decreased H3K4me3 and H3K27ac. (E) Integrative Genomics Viewer tracks showing up-regulated Phkg2 integrating with H3K4me3 and H3K27ac signals in GLA sperm. (F) Integrative Genomics Viewer tracks showing down-regulated Hsd17b11 integrating with H3K4me3 and H3K27ac signals in GLA sperm. Pink box indicates regions with differential H3K4me3 signals (P-value < 0.05). Blue box indicates regions with differential H3K27ac signals (P-value < 0.05). Dashed box indicates regions with slightly differential histone methylation signals.
FIGURE 5
FIGURE 5
Differential gene expression in 4-cell embryos in connection with H3K4me3 and H3K27ac alterations in GLA sperm. (A) Heatmap of normalized gene expression levels in CON and GLA 4-cell embryos. (B,C) Selected significant pathways from Gene Ontology analysis on up-regulated (B) or down-regulated (C) genes in GLA 4-cell embryos. (D) Venn diagram showing the number of regions with different gene expression levels in GLA 4-cell embryos that overlap regions with altered H3K4me3 and H3K27ac levels in GLA sperm. Left, up-regulated regions overlapping regions with increased H3K4me3 and H3K27ac. Right, down-regulated regions overlapping regions with decreased H3K4me3 and H3K27ac. (E) Integrative Genomics Viewer tracks showing up-regulated Mgl2 in 4-cell embryos integrating with H3K4me3 and H3K27ac signals in GLA sperm. (F) Integrative Genomics Viewer tracks showing down-regulated Dcn integrating with H3K4me3 and H3K27ac signals in GLA sperm. Blue box indicates regions with differential H3K27ac signals (P-value < 0.05). Dashed box indicates regions with slightly differential histone methylation signals.
FIGURE 6
FIGURE 6
Differentially parental-origin gene expression in 4-cell embryos in connection with H3K4me3 and H3K27ac alterations in GLA sperm. (A) Heatmap of normalized mRNA transcripts of paternal alleles in CON and GLA 4-cell embryos. (B) Integrative Genomics Viewer tracks showing RNA transcripts of parental-specific alleles (Usp36 and Zfp81) in 4-cell embryos integrating with H3K4me3 and H3K27ac signals in GLA sperm. (C) Heatmap of normalized mRNA transcripts of maternal alleles in CON and GLA 4-cell embryos. (D) Integrative Genomics Viewer tracks showing RNA transcripts of parental-specific alleles (Anxa1 and Sorl1) integrating with H3K4me3 and H3K27ac signals in GLA sperm. Dashed box indicates regions with slightly differential histone methylation signals.
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