Dual roles of mitochondrial akt in granulosa cells during ovarian folliculogensis and development of metabolic syndrome

Activating PI3K/Akt pathway triggers translocation of activated AKT into mitochondria, and granulosa cells are enriched with mitochondria. The goals of this study were to determine the mechanistic roles of mitochondrial AKT in granulosa cells during folliculogenesis and progression of menstruation cycles, and to explore its role in the regulation of whole body metabolism. To simulate ovarian insulin resistance in mitochondria, we have generated a novel transgenic mice model with Cre-LoxP system to disrupt mitochondrial AKT signaling in granulosa cells. The transgenic mice express a dominant negative mitochondria-targeting AKT in the granulosa cells (ovdnAKT). We used histological analysis, biochemical assays, and scRNA-seq to investigate folliculogenesis. Body composition was determined by EchoMRI and glucose homeostasis by glucose tolerance test. Insulin stimulates AKT phosphorylation and translocation to the mitochondria in granulosa cells in vivo. When mitochondrial AKT was inhibited in granulosa cells in 8-week old ovdnAKT mice, menstrual cycle became irregular and prolonged (ovdnAKT, 11.9 ± 3.7 days; controls, 4.5 ± 0.5 days, p<0.001). Vaginal lavage showed predominant diestrus phase in ovdnAKT mice. The ovary weight was increased (ovdnAKT, 10.19 ± 0.89 mg; controls, 6.28 ± 2.40 mg, p=0.049). Histology analysis revealed increased total number of follicles (ovdnAKT, 533.00 ± 14.00; controls, 337.67 ± 53.38, p=0.001) and increased accumulation of preantral follicles in ovdnAKT mice (ovdnAKT, 438.67 ± 22.59; controls, 284.33 ± 41.50, p=0.002). But morphologically mature antral follicles were nearly absent in the ovdnAKT mice. Ovary scRNA-seq cluster annotation confirmed an increased pool of preantral granulosa cells in the ovdnAKT ovary, decreased number of mature granulosa cells and atretic granulosa cells, and increased number of luteinizing granulosa cells. The population of mesenchymal cells, immune cells, epithelial cells, theca cells, and endothelial cells were not changed. Transcriptional analysis in the granulosa cells at preantral phase showed altered folliculogenesis-promoting and steroidogenesis genes in the ovdnAKT mice. Mitochondrial AKT also modulated whole body metabolism, body weight increased by 20%, fat mass/weight ratio increased by 83%, and lean mass/weight ratio decreased by 11% in the ovdnAKT mice after 16 weeks, indicating development of obesity and metabolic syndrome. Insulin stimulated translocation of active AKT to mitochondria in granulosa cells. Mitochondrial AKT played a critical role in the recruitment and maturation of antral follicles through transcriptional modulation. Disrupting this pathway in granulosa cells led to obesity and metabolic syndrome, which suggests ovary-adipose metabolic crosstalk.