The RNA content of sperm is implicated in the transmission of obesity from the paternal linage. Relative to their size mature sperm contain significant amounts of RNA including non-coding RNAs (ncRNAs), pseudogenes (processed/unprocessed) and protein coding mRNA, with sperm RNA content delivered to the oocyte at fertilisation. To date, only one paper has reported the variations in RNA content of sperm from obese men (1), focusing on small ncRNAs. Utilising bioinformatics pipelines and broad range RNA capture, we assessed the effects of obesity on human sperm RNA content, including those RNAs present/absent due to obesity.
RNA content was extracted using the Trizol method from purified motile sperm (swim up) from eight normal weight (BMI: 18-25kg/m2) and eight obese (BMI: >30kg/m2) Caucasian normospermic men (<45 years) undergoing infertility treatment due to female factor. Samples were processed with Bioo small RNA isolating kit (up to 150nt) and RNA-seq libraries sequenced with ~15 million reads. Raw data was analysed via two pipelines: standard bulk RNA-seq and the bcbio small RNA-seq. The limma-voom method identified differential gene expression.
The most abundant sperm RNA species were ribosomal RNA ~40%, mRNA ~25%, mitochondrial transfer RNAs ~9%, long ncRNAs ~7% and pseudogenes ~5%. 7452 RNAs in sperm were increased in abundance due to obesity (3106: 42% pseudogenes) and 160 decreased in abundance (33: 20% pseudogenes). 267 RNAs were unique to sperm from normal weight men (122: 46% pseudogenes) while only three RNAs were unique to sperm from obese men.
Pseudogenes contributed to the majority of differentially abundant sperm RNAs between normal weight and obese men. Previously presumed to lack function, evidence suggests that pseudogenes play important biological roles including initiating innate immune response, source of ncRNAs, DNA mediated regulation, protein transcription, and may also somewhat mediate the transmission of obesity from fathers to children.