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

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. MTX is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, however, the effect of paternal MTX exposure on their offspring has been largely unexplored. Moreover, paternal experiences can alter the health of the offspring through small RNAs such as miRNAs and tRNAs. To evaluate how methotrexate can affect the development of cranial cartilages on the offspring, we performed intraperitoneal injection of MTX in the Japanese medaka fish Oryzias latipes (males) and performed in vitro fertilization using sperm from treated males and oocytes from naive females (which had no contact with males) and evaluated the development of cranial cartilages such as basihyal and ceratohyal in their offspring. Moreover, we evaluated the abundance of miRNAs and tRNAs in the sperm of treated males by using small RNA-seq. Our results showed that several cartilages on the offspring were affect both at their length and their morphology. Additionally, the small RNA-seq showed that specific 5' tRNAs halves were significantly increased due to MTX treatment. Finally, UHPLC-MS-MS demonstrated that different post-transcriptional modification were altered in small RNAs isolated from the sperm of treated males. Our data suggests that paternal MTX-exposure influenced sperm tRNA methylation, as a result of alterations in the expression of certain RNA methyltransferases. These epitranscriptomic changes may cause the selective tRNA cleavage and the maintenance of certain 5' tRNA halves. These changes in the sperm RNA content and modifications might affect transcriptional cascades in the fertilized oocyte, with possible implications in cranial cartilage formation. The understanding of how tRNA modifications and their derived fragments impact on the transcriptional cascades occurring during early embryo will provide valuable insights into several diseases and it is expected that this will be a main focus of research in this field in the near future