Alpha-ketoglutarate enhances the proliferation and steroidogenic capacities of Tan sheep granulosa cells through autophagy pathway regulation.

Alpha-ketoglutarate（AKG）, a crucial intermediate in the tricarboxylic acid cycle, may affect ovarian function, oocyte maturation, and embryonic development in mammals. However, its specific impact on hormone secretion by granulosa cells remains undefined.In this study, we employed EdU, CCK-8, qRT-PCR, Western blot, and immunofluorescence analyses to observe that AKG can modulate the function and steroid metabolism of ovarian granulosa cells in Tan sheep. Our findings indicate that treatment with 5 mM AKG significantly stimulated granulosa cell proliferation, with up-regulated expression of Proliferating Cell Nuclear Antigen(PCNA) protein and mRNA, and an increased proportion of EdU positive cells. Concurrently, AKG enhanced cell survival by reducing the expression of the pro-apoptotic protein BAX, increasing the expression of the anti-apoptotic protein BCL2, and inhibiting pro-caspase3 activation. Additionally, AKG significantly increased the expression of key steroidogenic enzymes (CYP11A1, CYP19A1) and the secretion of estradiol and progesterone.Further investigation revealed that AKG attenuated autophagy by regulating the expression of autophagy related proteins (LC3II/I, ATG7, Beclin1, P62), reducing MDC fluorescence signals, and enhancing mitochondrial membrane potential.Experiments using rapamycin, an mechanistic target of rapamycin pathway inhibitor, confirmed that AKG regulates autophagy via activation of the mechanistic target of rapamycin signaling pathway, thereby influencing steroid hormone synthesis.In conclusion,this study is the first to reveal the molecular mechanisms by which AKG regulates ovarian function, AKG optimizes granulosa cell function by promoting proliferation, inhibiting apoptosis and autophagy, and enhancing steroid hormone synthesis, thereby providing a theoretical framework for improving follicle development and female animal fertility.