P-558 Childhood exposure to high-fat diets changes sperm small RNA content up to two unexposed generations of mice
Authors: Crisóstomo L , Bourgery M , Kotaja N , Raposo J F , Batterham R L , Alves M G 
Published: June 2022

Abstract

Study question
Is the adoption of high-fat diet from childhood associated with sperm small RNA content in mice? Are those changes inherited by the offspring and grand-offspring?

Summary answer
The adoption of high-fat diet during childhood changes sperm small RNA content up to the grand-offspring even after dietary correction.

What is known already
The prevalence of childhood obesity is increasing worldwide. The adoption of high-fat diet (HFD) from early age is a major factor for this trend. However, the long-lasting health effects of childhood obesity associated to HFD is unknown, particularly after dietary reversion. Multiple studies demonstrated that sperm small non-coding RNA (sncRNA) content changes in response to acute and chronic dietary factors. Also, sperm sncRNAs can transmit acquired traits such as metabolic syndrome. However, it is not known how the HFD-related sperm sncRNA fingerprints varies across generations, and how it relates with the phenotypes of the progeny.

Study design, size, duration
At weaning, 36 C57BL/6 mice were randomly divided into three groups (n = 12) feeding on 1) standard chow (CTRL) for 200 days, 2) HFD for 200 days, or 3) HFD for 60 days reverted to chow for 140 days (childhood HFD, CHFD). Subsequent generations (36 mice/generation, all fed on chow) were obtained by mating F0 and F1 males with same-age normoponderal females 120 days post-weaning and were assigned to the same experimental group as the progenitor.

Participants/materials, setting, methods
Mice were euthanized 200 days after weaning and tissues collected. Epidydimal sperm was collected, and concentration, viability, motility, and morphology were evaluated. Sperm RNA were extracted using the tri-reagent protocol, and sncRNAs sequenced by NGS RNA-seq. Sequences were annotated using SPORTS 1.1 and differential expression analysis performed using DESeq2. Targets of differently expressed sequences were estimated and Gene Ontology (GO) analysis of biological function performed using topGO.

Main results and the role of chance
HFD consumption during childhood is associated with poorer sperm parameters, even after dietary correction. No changes in sperm parameters were found between the offspring of HFD, CHFD and CTRL but, the grand-offspring of HFD and HFDt mice had lower sperm counts than the grand-offspring of CTRL. Regarding sperm sncRNA content, no changes were found between HFD and CTRL, but several mitochondrial transcription initiation RNAs (tiRNA) and repeat-derived RNAs (repRNAs) were differently expressed comparing to CTRL. Sperm sncRNA content of the offspring of HFD (piwi-interacting RNAs and repRNA) and CHFD (tRNA-derived fragments and repRNAs) displayed several differently expressed sequences comparing to CTRL. Only microRNAs (miRNAs) were differently expressed in the grandoffspring (CTRL vs. CHFD). GO analysis of biological function based on the targets of differently expressed sncRNAs showed a potential impact of childhood obesity in pathways related to sperm motility, chemotaxis and membrane integrity. These results suggest that sperm sncRNA content is altered due to childhood obesity even after two generations. This signature is not preserved across the male germline, but may influence the sperm sncRNA content of the progeny. This long-lasting, transgenerational signature of childhood obesity may explain the inheritance of the poor sperm quality phenotype from grand-sire to grand-offspring.

Limitations, reasons for caution
This model does not evaluate the impact of earlier dietary intervention, before sexual maturation. Also, it is not clear if the reported effects are exclusively due to childhood obesity or to excessive caloric intake. The estimates of biological impact using GO analysis of sperm sncRNA targets require further experimental evidence.

Wider implications of the findings
Our findings evidence the long-lasting effects of childhood obesity to sperm parameters, even after dietary reversion, and the potential transgenerational effects to the health of the progeny. Our results raise awareness about the impact of childhood obesity to reproductive health of future fathers and their progeny.

Trial registration number
0421/000/000/2016