P-682 Malic enzyme 1 in granulosa cells promotes ovarian aging through inhibiting the AMPK-FoXO1-IDH2 signaling pathway

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Abstract Study question Reduced levels of oxidative phosphorylation in granulosa cells are a key factor in regulating ovarian aging, but the upstream regulatory mechanisms are unknown. Summary answer Our study revealed that elevated malic enzyme 1 in granulosa cells promotes ovarian aging by inhibiting oxidative phosphorylation levels through the AMPK-FoXO1-IDH2 signaling pathway. What is known already With ovarian aging, the ovarian granulosa cells appear hallmarks of high apoptosis activity and low-proliferative activity, which lead to a reduction in ovarian reserve and a decrease in hormone synthesis, resulting in female infertility and reduced quality of life. And the decline of isocitrate dehydrogenase, IDH, in non-human primate ovarian granulosa cells is a key causative factor for the decline in the level of oxidative phosphorylation in granulosa cells. Recently, researchers reported that malic enzyme 1, which encodes a cytosolic, NADP-dependent Enzyme that generates NADPH for fatty acid biosynthesis inhibits IDH2 expression in tumor cells. Study design, size, duration Utilizing clinical samples, cells and animal models, with the help of research tools such as biochemical experiments, gene editing and single-cell transcriptome sequencing, at different levels, including molecular, cellular, animal and human samples, we systematically analyzed the characteristic changes and key molecules in granulosa cells along with ovarian aging and reveal the intracellular molecular pathways through which ME1 and IDH2 regulate the process of ovarian aging for 1 year. Participants/materials, setting, methods We first apply single-cell transcriptome sequencing analysis of young and old mouse ovaries to reveal cellular subpopulation changes and transcriptomic hallmarks of ovarian granulosa cell with aging. And then we validate the biological function and revealing the molecular mechanism to regulate ovarian aging by using physiological ovarian aging mouse model, clinical ovarian granulosa cells from young and old female and human ovarian granulosa cell line KGN utilizing technologies of protein immunology, qPCR and CRISPR/Cas9. Main results and the role of chance Ovarian single-cell transcriptome sequencing data from young and old mice showed that ovarian aging was accompanied by a dramatic decrease in the number of granulosa cells and drastic changes in the proportions of different subpopulations. The proportion of mitotically active granulosa cells was significantly lower in aging ovaries than in young mouse ovaries, and GO enrichment analysis showed that granulosa cell cycle, antioxidant and hormone synthesis-related signaling pathways were significantly down-regulated in aging ovaries. Using immunoblotting, immunofluorescence and qPCR, we demonstrated that the expression of ME1 in granulosa cells of aging mice and human ovaries was significantly elevated, while the expression of IDH2 was significantly decreased. Performing the overexpression and knockdown of ME1 in human ovarian granulosa cell line KGN, which further demonstrated that ME1 could inhibit the transcriptionally regulated IDH2 expression of downstream genes, such as FoXO1, by suppressing the phosphorylation of AMPK. The decrease of IDH2 in mitochondria reduces the level of oxidative phosphorylation in granulosa cells, resulting in the increase of intracellular reactive oxygen species, activation of the intracellular p53-p21 signaling pathway, inhibition of the cell cycle, and promotion of apoptosis, which in turn impairs the function of granulosa cells to synthesize hormones and promotes ovarian aging. Limitations, reasons for caution Malic enzyme 1, which links the glycolytic cycle to the citric acid cycle, expressing in other ovarian cells including oocytes, and the regulation of its expression is complex. Future studies will focus on its biological functions in other ovarian cells and its molecular mechanism in regulating ovarian aging. Wider implications of the findings Our study suggests that granulosa cell ME1 and IDH2 co-regulate oxidative phosphorylation levels to regulate ovarian aging, and that ME1 and IDH2 may serve as potential targets for the finding and treatment of premature ovarian aging and delaying ovarian aging in women. Trial registration number not applicable