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. 2021 Sep;20(9):e13466.
doi: 10.1111/acel.13466. Epub 2021 Aug 27.

Altered sperm tsRNAs in aged male contribute to anxiety-like behavior in offspring

Yi Guo  1   2 , Dandan Bai  2 , Wenqiang Liu  2 , Yingdong Liu  2 , Yalin Zhang  2 , Xiaochen Kou  2 , Jiayu Chen  2 , Hong Wang  2 , Xiaoming Teng  2 , Ji Zuo  1 , Shaorong Gao  2
Affiliations

Altered sperm tsRNAs in aged male contribute to anxiety-like behavior in offspring

Yi Guo et al. Aging Cell. 2021 Sep.

Abstract

Parental age at first pregnancy is increasing worldwide. The offspring of aged father has been associated with higher risk of several neuropsychiatric disorders, such as schizophrenia and autism, but the underlying mechanism remains elusive. Here we report that advanced paternal age in mice alters the profile of transfer RNA-derived small RNAs (tsRNAs). Injection of sperm tsRNAs from aged male mice into zygotes induced anxiety-like behaviors in F1 males. RNA sequencing of the cerebral cortex and hippocampus of those F1 male mice altered the gene expression of dopaminergic synapse and neurotrophin. tsRNAs from aged male mice injection also altered the neuropsychiatry-related gene expression in two-cell and blastocyst stage embryos. More importantly, the sperm tsRNA profile changes significantly during aging in human. The up-regulated sperm tsRNA target genes were involved in neurogenesis and nervous system development. These results suggest that aging-related changes of sperm tsRNA may contribute to the intergenerational transmission of behavioral traits.

Keywords: Advanced paternal age; anxiety; offspring; pre-implantation embryo; sperm; transfer RNA-derived small RNA.

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Conflict of interest statement

Yi Guo, Dandan Bai, Wenqiang Liu, Yingdong Liu, Yalin Zhang, Xiaochen Kou, Jiayu Chen, Hong Wang, Xiaoming Teng, Ji Zuo, Shaorong Gao declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Shanghai First Maternity and Infant Hospital, Shanghai, China) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.

Figures

FIGURE 1
FIGURE 1
Behavioral traits in F1 mice arising from natural mating or from zygotes injected with total sperm RNAs from aged or young mice. (a) Schematic of the potential role of sperm RNAs in mediating hereditary transmission of anxiety‐like behavior to F1 male mice. (b‐d) Behavioral tests for F1 male offspring (n = 10 for each group) obtained by naturally mating female mice 10 weeks old with male mice 14–18 months old (aged male mice, Ma) or male mice 3–4 months old (young male mice, My). (b) Evaluation of anxiety‐like behavior in the open field test. (c) Evaluation of social ability in the three‐chambered social interaction test. (d) Evaluation of stereotypic behavior in the marble‐burying test. (e‐h) F1 male mice (n = 10 for each group) arising from zygotes injected with total sperm RNAs from Ma or My groups. (e) Scheme of injection of sperm total RNA into normal zygotes and growth curves of the resulting F1 male offspring. (f) Evaluation of anxiety‐like behavior in the open field test. (g) Evaluation of anxiety‐like behavior in the elevated plus‐maze test. (h) Evaluation of social ability in the three‐chambered social interaction test. *p < 0.05; **p < 0.01; ns, not significant
FIGURE 2
FIGURE 2
Aging alters the sperm tsRNA profile in mice. Sperm tsRNAs from aged male mice (Ma) and young male mice (My) were detected using Small RNA‐seq. (a) Distribution of read lengths. (b) Heat map of sperm tRNA‐derived fragments (tRFs and tiRNAs) differentially expressed between the Ma group and My group. (c) Volcano plot of sperm tRFs and tiRNAs differentially expressed between the Ma group and My group. (d) KEGG pathway analysis of differentially expressed tRFs and tiRNAs that were up‐regulated in the Ma group. Data are from n = 3 independent samples for the Ma group and n = 4 for the My group
FIGURE 3
FIGURE 3
Sperm tsRNA from aged mice is associated with anxiety‐like behavior in F1 male mice. (a) Schematic of the procedure for evaluating the potential role of sperm tsRNA in hereditary transmission of anxiety‐like behavior to F1 male mice. F1 male offspring were obtained from zygotes that had been injected with sperm tsRNAs from aged male mice (Ma) or young male mice (My) (n = 10 mice for each group). (b) Evaluation of anxiety‐like behavior in the open field test. (c) Evaluation of anxiety‐like behavior in the elevated plus‐maze test. (d) Heat map of genes differentially expressed between the Ma group and My group in the cerebral cortex and hippocampal tissue. (e) KEGG enrichment analysis of differentially expressed genes in the cerebral cortex and hippocampal tissue. (f) Validation of some differentially expressed genes based on quantitative PCR. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ns: not significant
FIGURE 4
FIGURE 4
Sperm tsRNAs from aged mice dysregulate gene expression in pre‐implantation embryos. (a) Schematic of how gene expression was analyzed in pre‐implantation embryos. (b) Heat map of DEGs from blastocysts from the aged mouse (Ma) group and the young mouse (My) group. (c) Venn diagrams showing the intersection of genes targeted by differentially expressed tsRNAs and differentially expressed genes in blastocysts. KEGG pathway analysis of genes up‐ or down‐regulated in the Ma group relative to the My group. (d) Hierarchical cluster analysis (heat map) of results from two‐cell embryos from the Ma group and My group. (e) Venn diagrams showing the intersection of genes targeted by differentially expressed tsRNAs and differentially expressed genes in two‐cell embryos. Data are from n = 3 independent samples for each group
FIGURE 5
FIGURE 5
Aging alters the sperm tsRNA profile in men. Sperm tsRNAs from aged male human (Ha) and young male human (Hy) were detected using Small RNA‐seq. (a) Principal Component Analysis (PCA) on RNA‐Seq data from the Ma group and My group. (b) Distribution of read lengths. (c) Heat map and volcano plot of sperm tRNA‐derived fragments (tRFs and tiRNAs) differentially expressed between the Ma group and My group. (d)Venn diagram based on number of specifically expressed tRFs and tiRNAs. (e) Distribution of tRF and tiRNA subtypes. The numbers in brackets indicate the number of tRF or tiRNA in that subtypes. (f) Enrichment of gene ontology (GO) terms among tRFs and tiRNAs up‐regulated in Ha relative to Hy. Data are from n = 3 independent samples for each group
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