Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs

Abstract

There is a growing body of evidence linking maternal overnutrition to obesity and psychopathology that can be conserved across multiple generations. Recently, we demonstrated in a maternal high-fat diet (HFD; MHFD) mouse model that MHFD induced enhanced hedonic behaviors and obesogenic phenotypes that were conserved across three generations via the paternal lineage, which was independent of sperm methylome changes. Here, we show that sperm tRNA-derived small RNAs (tsRNAs) partly contribute to the transmission of such phenotypes. We observe increased expression of sperm tsRNAs in the F1 male offspring born to HFD-exposed dams. Microinjection of sperm tsRNAs from the F1-HFD male into normal zygotes reproduces obesogenic phenotypes and addictive-like behaviors, such as increased preference of palatable foods and enhanced sensitivity to drugs of abuse in the resultant offspring. The expression of several of the differentially expressed sperm tsRNAs predicted targets such as CHRNA2 and GRIN3A, which have been implicated in addiction pathology, are altered in the mesolimbic reward brain regions of the F1-HFD father and the resultant HFD-tsRNA offspring. Together, our findings demonstrate that sperm tsRNA is a potential vector that contributes to the transmission of MHFD-induced addictive-like behaviors and obesogenic phenotypes across generations, thereby emphasizing its role in diverse pathological outcomes.

Keywords: epigenetic; maternal; obesity; overnutrition; sperm RNA.

Fig. 1.

Breeding scheme to generate sperm sncRNA-injected offspring. Female mice (F0) were fed HFD or chow diet for 3 wk preconception and 6 wk during gestation and lactation to obtain F1-HFD and F1-CTR offspring, respectively. Mature sperm was isolated from F1-HFD and F1-CTR males at PND 70. Sperm total RNAs from F1-HFD and F1-CTR males were separated on a 6% TBE gel. Sperm total RNAs as well as isolated RNA fragments at sizes ∼70–90 nt, ∼40–45 nt, and ∼30–34 nt were purified and microinjected into male pronuclei of normal fertilized eggs to generate total RNA, tRNA, T40RNA, and tsRNA offspring from both groups. Offsprings from HFD and CTR groups were on chow diet since weaning. HFD and CTR offspring groups refer to the descendants of 9 wk HFD- and chow-fed F0 dams, respectively. Colors of the mice are matched with the color codes used for the groups in subsequent graphs.

Fig. 2.

Altered metabolic phenotypes in HFD-total RNA–injected offspring. (A) Body weight: HFD-total RNA offspring showed gradually increased body weight compared with the CTR-total RNA offspring. CTR-total RNA, n = 19 (8 M, 11 F); HFD-total RNA, n = 18 (8 M, 10 F). (B) Insulin tolerance test: HFD-total RNA offspring had higher blood glucose level than CTR-total RNA following an insulin injection. HFD-total RNA male offspring showed a stronger impairment of insulin sensitivity. CTR-total RNA, n = 19 (8 M, 11 F); HFD-total RNA, n = 16 (8 M, 8 F). (C–G) Distribution of fat: HFD-total RNA offspring displayed a marked increase in total fat, s.c. fat, visceral fat, and fat mass ratio, with no difference in lean mass. CTR-total RNA, n = 17 (7 M, 10 F); HFD-total RNA, n = 14 (6 M, 8 F). (H–K) Plasma parameters: HFD-total RNA group showed higher fasted plasma insulin and cholesterol and lower FFA levels but no difference in plasma triglyceride (TG) levels compared with CTR (n = 6 M/6 F per group). Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). F, female; M, male.

Fig. 3.

Altered hedonic response to natural rewards in total RNA-injected offspring. (A) HFD preference: no difference in HFD consumption between HFD-total RNA and CTR-total RNA offspring was observed. (B) Sucrose preference: all offspring groups showed increased sucrose consumption at all concentrations. No difference was observed in sucrose preference between groups. HFD-total RNA, n = (7 M, 9 F); CTR-total RNA, n = (8 M, 11 F). Data are presented as mean ± SEM. F, female; M, male.

Fig. 4.

Altered hedonic response to drugs of abuse in total RNA-injected offspring. (A) Alcohol preference: HFD-total RNA offspring showed increased preference for alcohol compared with CTR. Male and female HFD-total RNA offspring consumed more alcohol at higher concentrations compared with their CTR littermates. HFD-total RNA, n = (7 M, 9 F); CTR-total RNA, n = (8 M, 11 F). (B) Amphetamine sensitivity: male and female HFD-total RNA offspring showed enhanced locomotor response to amphetamine (AMPH) compared with their CTR littermates (n = 5 M/5 F per group). Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). Base, baseline; Sal, saline. F, female; M, male.

Fig. 5.

Altered hedonic responses to natural rewards in sperm RNA fragment-injected offspring. HFD preference: (A) all offspring groups consumed more HFD than chow diet, and females consumed more than males. HFD-tsRNA offspring showed a greater intake of HFD than CTR-tsRNA offspring. HFD-tsRNA, n = 10 M/7 F; CTR-tsRNA, n = 10 M/10 F. (B) No difference was detected in HFD preference between the groups (n = 8 M/8 F per group). (C) No difference was observed in HFD preference between the groups (n = 8 M/8 F per group). Sucrose preference: (D) HFD-tsRNA offspring showed greater sucrose consumption than CTR-tsRNA offspring. Male HFD-tsRNA offspring showed a stronger sucrose preference compared with the others at all concentrations. HFD-tsRNA, n = 10 M/7 F; CTR-tsRNA, n = 10 M/10 F. (E) No difference in sucrose preference was detected between the tRNA offspring groups (n = 8 M/8 F per group). (F) No difference in sucrose consumption between the T40RNA offspring groups was detected (n = 8 M/8 F per group). Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). F, female; M, male.

Fig. 6.

Altered hedonic responses to drugs of abuse in sperm RNA fragment-injected offspring. Alcohol preference: (A) HFD-tsRNA offspring consumed more alcohol than CTR-tsRNA offspring. Female HFD-tsRNA offspring had stronger alcohol preference compared with the others at higher concentrations. HFD-tsRNA, n = 9 M/10 F; CTR-tsRNA, n = 7 M/6 F. (B) No difference in alcohol consumption between the groups was detected (n = 8 M/8 F per group). (C) HFD-T40RNA offspring did not differ in alcohol consumption at all concentrations compared with the CTR-T40RNA offspring (n = 8 M/8 F per group). Amphetamine sensitivity: (D) male and female HFD-tsRNA offspring showed enhanced locomotor response to amphetamine (AMPH) compared with the CTR-tsRNA offspring, with no difference in locomotion at baseline (Base) and following a saline solution (Sal) injection. (E) HFD-tRNA offspring did not differ in baseline locomotor activity following a saline solution injection and after amphetamine challenge compared with the CTR-tRNA offspring. (F) No difference was observed in baseline spontaneous locomotor activity and after a saline solution injection as well as after an amphetamine challenge between the T40RNA offspring groups (n = 5 M/5 F per group). Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). F, female; M, male.

Fig. 7.

Altered metabolic phenotypes in HFD-tsRNA offspring following junk food choice test. (A) Experimental design: tsRNA offspring from HFD and CTR groups were given free access to HFD and 1% sucrose along with chow and water for 12 wk, starting 2 wk after weaning (at postnatal week 5). (B) Body weight: HFD-tsRNA offspring gradually gained more weight compared with the CTR littermates following junk food choice exposure. The weight gain was more marked in male HFD-tsRNA offspring compared with the others. CTR-tsRNA, n = 21 (12 M, 9 F); HFD-tsRNA, n = 23 (13 M, 10 F). (C) Insulin tolerance test: HFD-tsRNA offspring had persistently higher blood glucose level than CTR-tsRNA offspring following insulin injection. A stronger impairment of insulin sensitivity was detected in male HFD-tsRNA offspring. CTR-tsRNA, n = 17 (9 M, 8 F); HFD-tsRNA, n = 22 (11 M, 11 F). (D) Food intake: male and female HFD-tsRNA offspring showed marked increase in HFD consumption over the weeks compared with the CTR-tsRNA offspring. (E) Solution intake: male and female HFD-tsRNA offspring consumed more sucrose solution compared with the CTR-tsRNA offspring. No difference was observed in chow food and water consumption between the groups. CTR-tsRNA, n = 20 (12 M, 8 F); HFD-tsRNA, n = 23 (13 M, 10 F). Data are presented as mean ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001). F, female; M, male.

Fig. 8.

Sperm sncRNA profile in F1 fathers and differences in transcriptomes in brain of F1 father and tsRNA offspring. (A) Length distribution of sncRNA sequence reads from F1-HFD and F1-CTR males. Error bars indicate SD. (B) Principal component analysis based on 4,822 differentially expressed sncRNA species. x and y axes show principal components 1 (PC1) and 2 (PC2) that explain 83.9% and 5.6% of the total variance, respectively. (C) Composition of sncRNA transcriptomes. tsRNAs constitute the majority of sncRNAs in HFD and CTR groups. (Inset) Example of relative sncRNA coverage of tRNA-Gly-GCC-2–8. Error bars indicate SD. (D) Heat map displays relative expression of tsRNAs derived from different tRNAs across HFD and control probes. Thirteen tRNA fragments showed a significant difference in expression. Clustering bases on average linkage. The Pearson distance measurement method was used. (E) The increased level of CHRNA2 in the dSTR of F1 HFD and HFD-tsRNA offspring compared with CTR littermates. (F) Decreased levels of CHRNA2 in the Nac of F1-HFD and HFD-tsRNA offspring compared with their CTR littermates. (G) Higher expression of CHRNA2 in the VTA of F1-HFD male and HFD-tsRNA offspring compared with CTR. F1-CTR, n = 6; F1-HFD, n = 6; CTR-tsRNA, n = 12; HFD-tsRNA, n = 12. Data are presented as mean ± SEM (*P < 0.05).