Demystifying the functions of the mitochondrial ORFan proteins in bivalves with doubly uniparental inheritance

Strict maternal inheritance of mitochondria is known to be the rule in animals, but over 100 species across six orders of bivalves possess doubly uniparental inheritance (DUI) of mitochondria. Under DUI, two distinctive sex-specific mitogenomes coexist. In marine and freshwater mussels, each mitogenome has an additional protein-coding gene, called female- and male-specific open reading frame or forf and morf, respectively. The function(s) of the associated FORF and MORF proteins remain unknown. Herein, we show that these proteins present similar tissue expression patterns in two distantly related DUI species: MORF was only expressed in male gonads, whereas FORF was expressed in all tissues of both sexes in the marine mussel Mytilus edulis and the freshwater mussel Venustaconcha ellipsiformis. Moreover, MORF was only expressed during the reproductive season, while FORF presented no clear seasonality pattern in M. edulis. Immunocytochemistry revealed the presence of both proteins in mitochondria and acrosomes of late spermatids and mature sperm. We hypothesize that MORF has a key function in spermatogenesis, while FORF has a more general function in both sexes. We also propose that both proteins may be involved in the fertilization process. The involvement of MORF in paternal mitochondrial transmission is also discussed.