Exploring the potential of Schmidtea mediterranea as a model for reproductive toxicity through analysis of sterility caused defective m6A RNA regulation

Development of alternative methods for analysis of reproductive toxicity is a challenge that requires the characterization of mechanistic endpoints linked to the toxicity. <i>N</i> ⁶-methyladenosine (m⁶A) is one of the most prevalent post-transcriptional modifications of eukaryotic RNA molecules and one that supports development of gametes in mammals. While the role of m⁶A RNA regulation in the soma of planarian flatworms has been previously studied, the presence and biological relevance of this regulatory pathway in the germline of these or other lophotrochozoans remains unknown. Here, we characterize m⁶A RNA regulation factors in the planarian <i>Schmidtea mediterranea</i> and analyze their function in development of the germline. Enriched expression of orthologs of m⁶A methyltransferase complex components, namely <i>Smed-METTL3</i>, <i>Smed-METTL14</i>, and <i>WTAP</i>, was detected in ovaries and testes of <i>S. mediterranea</i>. Perturbation of these factors by RNA-interference (RNAi) disrupted intermediate steps in oogenesis and spermatogenesis, but the presence of germline stem cells was not affected. Expression of m⁶A "reader" homologs was also detected in the planarian gonads. Sperm development defects were observed upon <i>Smed-YTHDF2-3</i> RNAi, and combined knockdown of <i>Smed-YTHDF2-3, Smed-YTHDF2-2</i>, or <i>Smed-YTHDF1-2</i> with that of writers led to increased frequency of germline development defects. These results indicate that regulation of RNA by m⁶A methylation supports male and female germline development in the planarian <i>S. mediterranea</i> as it does in mammals. This study, along with preexisting examples of conserved genetic mechanisms in germline development, showcase planarians as a promising option for early screening steps in reproductive toxicity assays within the scope of male and female fertility.