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 (OVCH2), 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 OVCH2 influences sperm functionality. Deletion of Ovch2 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 of OVCH2 with sperm surface proteins critical for fertilization, highlighting both its essential protease domain and the transient, finely regulated nature of its substrates. Collectively, these findings position OVCH2 as an orchestrator of sperm surface remodeling, with broad implications for fertility diagnostics and therapeutic interventions. By revealing a multifaceted network of OVCH2-dependent pathways, this study provides a mechanistic framework for developing innovative strategies to counter idiopathic male infertility and to enhance male contraceptive design.