Small Molecule Ovastacin Inhibitors Preserve Fertilization Competence of Murine Eggs by Mimicking Fetuin-B Function

Infertility represents a major global health challenge and has driven increasing reliance on ART. However, fertilization success in vitro is often limited when physiological egg-sperm interaction is preserved, largely due to premature hardening of the zona pellucida (ZP), the extracellular matrix surrounding the oocyte that controls sperm access. ZP hardening is physiologically triggered after fertilization by ovastacin-mediated cleavage of zona pellucida protein 2 (ZP2) to protect the early embryo. However, if not inhibited prematurely, this cleavage reduces egg competence. In vivo, ovastacin is restrained by the plasma protein fetuin-B, largely absent in IVF media. Whether this process can be pharmacologically targeted has yet to be explored. Here, we identify and functionally characterize small molecule hydroxamate inhibitors that transiently inhibit ovastacin in murine oocytes and evaluate their ability to control fertilization competence. A lead compound preserved ZP2 integrity and fertilization rates at 86% (±1%) after 8 hours of in vitro culture, compared to 57% (±7%) in controls. Importantly, inhibitor-treated embryos developed to the blastocyst stage without abnormalities. These findings establish transient ovastacin inhibition as simple, non-invasive pharmacological strategy to ensure IVF outcomes while preserving physiological egg-sperm interaction mechanisms.