Elevated N‐glycosylated cathepsin L impairs oocyte function and contributes to oocyte senescence during reproductive aging

Abstract Age‐related declines in oocyte quality and ovarian function are pivotal contributors to female subfertility in clinical settings. Yet, the mechanisms driving ovarian aging and oocyte senescence remain inadequately understood. The present study evaluated the alterations in N‐glycoproteins associated with ovarian aging and noted a pronounced elevation in N221 glycopeptides of cathepsin L (Ctsl) in the ovaries of reproductive‐aged mice (8–9 months and 11–12 months) compared to younger counterparts (6–8 weeks). Subsequent analysis examined the involvement of Ctsl in oocyte aging and demonstrated a significant elevation in Ctsl levels in aged oocytes. Further, it was revealed that the overexpression of Ctsl in young oocytes substantially diminished their quality, while oocytes expressing an N221‐glycosylation mutant of Ctsl did not suffer similar quality degradation. This finding implies that the N221 glycosylation of Ctsl is pivotal in modulating its effect on oocyte health. The introduction of a Ctsl inhibitor into the culture medium restored oocyte quality in aged oocytes by enhancing mitochondrial function, reducing accumulated reactive oxygen species (ROS), lowering apoptosis, and recovering lysosome capacity. Furthermore, the targeted downregulation of Ctsl using siRNA microinjection in aged oocytes enhanced fertilization capability and blastocyst formation, affirming the role of Ctsl knockdown in fostering oocyte quality and embryonic developmental potential. In conclusion, these findings underscore the detrimental effects of high expression of N‐glycosylated Ctsl on oocyte quality and its contribution to oocyte senescence, highlighting it as a potential therapeutic target to delay ovarian aging and enhance oocyte viability.