Evidence That Heat Shock Protein A5 (HSPA5) Plays a Role During Bovine <i>In Vitro</i> Embryo Production

Oviductal fluid (OF) proteins, such as HSPA5, play essential roles in reproductive physiology, including zona pellucida (ZP)-sperm interactions. Hypothesizing that HSPA5 interacts with oocytes, we evaluated its effects during the last 4 h of bovine in vitro cumulus-oocyte complex (COC) maturation and subsequent embryo development. COCs were aspirated from slaughterhouse-derived cow ovaries and matured for 20 h in base medium (BM). The medium was then partially replaced with BM (control) or BM supplemented with recombinant HSPA5 (100 ng/ml) for the final 4 h of maturation. After maturation, COCs were either denuded for analysis or used for in vitro fertilization (IVF) and culture (IVC) to assess embryo development to the blastocyst stage. To investigate HSPA5's role, we assessed the mitochondrial membrane potential (MMP), reactive oxygen species (ROS), glutathione (GSH) content in COCs and maturation medium, zona pellucida (ZP) solubility, oocyte penetration, blastocyst rates, and gene expression. Recombinant HSPA5 (rHSPA5) enhanced oocyte mitochondrial activity (p = 0.0006) and significantly increased the blastocyst yield (p = 0.04). Blastocysts from rHSPA5-treated oocytes showed upregulation of 19 genes associated with embryo quality, DNA methylation, cell growth, development and oxidative stress. Although rHSPA5 did not alter the monospermic fertilization rate or oxidative stress in oocytes, it improved mitochondrial function, supported blastocyst development, and modulated embryonic transcriptional profiles. These findings suggest that rHSPA5 plays a beneficial role during the final phase of oocyte maturation, contributing to improved embryo quality and developmental competence in vitro.