Effects of CSTB on in vitro maturation of ovine oocytes

Cystatin B (CSTB) primarily acts as an intracellular cysteine cathepsin inhibitor and plays important biological functions in multiple tissues. This study aimed to investigate the expression of CSTB in ovine ovaries and its effect on the in vitro maturation of ovine oocytes. We cloned a 390 bp CSTB cDNA fragment, containing 297 bp coding sequence and encoding 98 amino acids. The amino acid sequence of the homologues of ovine CSTB is 72.45-98.98 % similar to other species. In addition, CSTB is highly expressed in the ovary and uterus of the reproductive system, specifically localized in granulosa cells and oocytes. Adding recombinant CSTB to in vitro maturation medium increased the maturation rate, cleavage rate and blastocyst rate of small follicle oocytes. Conversely, interfering with CSTB knockdown reduced the maturation rate and developmental potential of oocytes. Recombinant protein upregulated mitochondrial membrane potential, ATP, and autophagy protein LC3A/LC3B in oocytes while downregulated reactive oxygen species. In contrast, CSTB knockdown reversed these trends, resulting in significant downregulation of membrane potential, ATP, and LC3A/LC3B and upregulation of reactive oxygen species. In conclusion, CSTB is a critical functional molecule for the in vitro maturation of ovine oocytes. It regulates oocyte developmental potential by modulating reactive oxygen species (ROS), membrane potential and autophagy in ovine oocytes. These findings enhance the understanding of the role of CSTB in ovine oocyte maturation in vitro.