Ovochymase 2 is a key regulatory factor modulating proteolytic pathways and sperm maturation in the mammalian epididymis†.

Spermatozoa acquire fertilizing competence during epididymal transit through proteolytic, chaperone-mediated, and post-translational modifications. Ovochymase 2, an epididymis-specific trypsin-like serine protease, has emerged as a central regulator of this maturation process. Here, we integrate targeted gene disruption, comprehensive proteomic profiling, and affinity-based proteome enrichment to delineate how Ovochymase 2 influences sperm functionality. Deletion of Ovochymase 2 disrupts the proteome of epididymal sperm, resulting in diminished levels of core fertility-related factors-including a disintegrin and metalloprotease domain 3, β-defensins, and protease-inhibitor complexes-while inducing compensatory upregulation of alternate proteases and chaperones. Interaction assays confirm direct or indirect associations between Ovochymase 2 and sperm surface proteins critical for fertilization, highlighting its essential protease domain and the transient, and finely regulated nature of its substrates. Collectively, these findings position Ovochymase 2 as an orchestrator of sperm surface remodeling, with broad implications for fertility diagnostics and therapeutic interventions. By revealing a multifaceted network of Ovochymase 2-dependent pathways, this study provides a mechanistic framework for developing innovative strategies to counter idiopathic male infertility and to enhance male contraceptive design.