Loss of ACR and Retention of Its Ancient Paralog TMPRSS12 in the Evolution of Teleost Fertilization

Acrosin (ACR) and transmembrane protease serine 12 (TMPRSS12) are sperm-related S1/trypsin-like serine proteases involved in mammalian male fertility, but their evolutionary relationship in vertebrates remains unclear. Here, we combined phylogenetic, synteny, selection-pressure, and transcriptomic analyses to investigate their evolutionary origin and divergence. OHNOLOGS v2 identified ACR–TMPRSS12 as a candidate ohnolog pair with strong outgroup support, and OrthoFinder placed their duplication in the ancestor of jawed vertebrates. Phylogenetic analyses recovered ACR and TMPRSS12 as reciprocally monophyletic sister clades, consistent with an ancient duplication that may be associated with the two rounds of whole-genome duplication (2R-WGD). Their subsequent evolutionary trajectories differed markedly. ACR was retained in chondrichthyans, sarcopterygians, tetrapods, and acipenseriforms, whereas its widespread loss in other actinopterygians largely coincided with acrosome loss, although bichirs retained the acrosome but lacked ACR. TMPRSS12 was widely retained across jawed vertebrates but showed relaxed selection in Poeciliidae and independent losses in Sebastes and Takifugu. Both genes exhibited testis-biased transcript expression, and their proteins retained a relatively conserved trypsin-like catalytic domain. These findings reveal contrasting patterns of gene retention between ACR and TMPRSS12 and lineage-specific variation in selective constraints on TMPRSS12, consistent with their divergence following an ancient duplication event.