Autophagy is a fundamental process involved in regulating cellular homeostasis. Autophagy has been classically discovered as a cellular process that degrades cytoplasmic components non-selectively to produce energy. Over the past few decades, this process has been shown to work in energy production, as well as in the reduction of excessive proteins, damaged organelles, and membrane trafficking. It contributes to many human diseases, such as neurodegenerative diseases, carcinogenesis, diabetes mellitus, development, longevity, and reproduction. In this review, we provide important information for interpreting results related to autophagic experiments and present the role of autophagy in this field.

Aging drives excessive ovarian oxidative stress (OS), impairing fertility and affecting granulosa cells (GCs), which are involved in folliculogenesis. This study aims to clarify the relationship between OS and autophagy in GCs and to identify compounds that enhance OS resistance. We identified Rubicon, an autophagy suppressor, as a key mediator of DNA damage in GCs under OS. Hydrogen peroxide (H₂O₂) compromised cell viability via DNA damage in the human GC cell line, HGrC1, without affecting autophagic activity. However, autophagy activation increased OS resistance in HGrC1 cells, and vice versa. Among clinically safe materials, trehalose, a disaccharide, protected cells as an autophagy activator against H₂O₂-induced cytotoxicity. Trehalose significantly increased autophagic activity, accompanied by reduced Rubicon expression, compared to other carbohydrates. It also reduced the expression of DNA damage-responsive proteins and the production of reactive oxygen species. Rubicon knockdown mitigated OS-induced DNA damage, while Rubicon overexpression enhanced DNA damage and decreased HGrC1 cell viability. Trehalose enhanced OS resistance by activating autophagy and suppressing Rubicon in a bidirectional manner. As Rubicon expression increases in aged human ovaries, trehalose may improve ovarian function in patients with infertility and other OS-related diseases.

Aging increases oxidative stress (OS) in the ovaries, leading to infertility-related dis-orders. Granulosa cells (GCs), which support folliculogenesis in the ovaries, are chron-ically exposed to excessive OS under pathological conditions. Here, we demonstrate the significant role of Rubicon, an autophagy suppressor, as a DNA damage mediator in GCs under OS. Hydrogen peroxide (H2O2) decreased cell viability in the human GC cell line, HGrC1, due to DNA damage, without affecting autophagic activity. However, activation of autophagy increased OS resistance in HGrC1 cells, and vice versa. Among several materials that are considered safer for clinical use, trehalose, a disaccharide, exerted a protective effect as an autophagy activator against H2O2-induced cytotoxici-ty. Trehalose significantly increased autophagic activity, accompanied by a reduction in Rubicon, compared to other carbohydrates. It also reduced the expression of DNA damage-responsive proteins and the production of reactive oxygen species. The reduc-tion in Rubicon expression by siRNA mitigated DNA damage caused by OS, while overexpression of Rubicon enhanced DNA damage and decreased HGrC1 cell viability. Trehalose enhanced OS resistance by activating autophagy and suppressing Rubicon in a bidirectional manner. As Rubicon expression increases in aged human ovaries, tre-halose may improve ovarian function in patients with infertility and other OS-related diseases.