Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring

Abstract

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5’ tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

Prevention of transgenerational transmission of disease susceptibility through perinatal intervention

“…However, it has become clear that a set of functional RNAs exists and has been characterized in mature sperm that are delivered to the oocyte at fertilization and contribute to early embryonic development, thus influencing the phenotypic outcome of the offspring [27]. Small noncoding RNAs (sncRNAs) are potential carriers of non-genetic information in sperm [28,29], and tRNA-derived small RNAs (tsRNAs) and miRNAs are most abundant in mature sperm. Sperm tsRNAs have been identified as molecular mediators of paternal experiences such as high-fat diets [30,31], lowprotein diets [32], and stress [33].…”