Activation of mitochondrial CPS1 promotes dormant ovarian follicle activation via arginine elevation and the mTORC1 pathway

Background: Carbamoyl phosphate synthetase 1 (CPS1) is a mitochondrial ligase essential for urea production, catalyzing the formation of carbamoyl phosphate from ammonia in a reaction requiring the allosteric activator N acetyl L glutamate (NAG). Through the urea cycle, CPS1 activity ultimately contributes to arginine synthesis, a known activator of mTOR. The NAG analogue N-carbamoyl-L-glutamate (NCG) has been shown to promote follicle development in several species, potentially via mTOR signaling; however, its specific role in folliculogenesis remains unclear. Methods: Cultured murine ovaries and human ovarian cortical tissue fragments were treated with NCG to assess its effects on primordial follicle activation. Human granulosa like KGN cells were used to evaluate NCG induced changes in proliferation, arginine levels, and mTOR pathway activation. Mechanistic studies examined the interaction between CASTOR1 and GATOR2 in response to NCG supplementation. Results: NCG mediated activation of CPS1 promoted the awakening of dormant (primordial) follicles in both mouse ovaries and human ovarian cortical tissues. In KGN cells, NCG enhanced cell proliferation and increased intracellular arginine levels, accompanied by an elevated pS6K/S6K ratio, consistent with mTORC1 activation. Mechanistically, NCG induced arginine accumulation reduced CASTOR1 binding to its inhibitor GATOR2, thereby facilitating S6K phosphorylation and activation of the mTORC1 signaling cascade. siRNA-mediated knockdown reduced CPS1 protein levels, which eliminated NCG's effect on arginine levels and the phosphorylation of S6K. Conclusion: NCG activates CPS1 to elevate arginine levels, relieve CASTOR1 mediated inhibition of GATOR2, and stimulate mTORC1 signaling. This pathway promotes primordial follicle activation and supports follicle development, highlighting NCG as a potential modulator of ovarian folliculogenesis.