A Preliminary Study Of Geranylgeranyl Diphosphate Synthase(GGPPS)in Regulating Premature Ovarian Failure

Premature ovarian failure(POF)is a common disease characterized by amenorrhea in women under the age of 40,with elevated levels of low estrogen and gonadotropins and high infertility rate.Early screening and diagnosis have guiding value for conception or oocyte preservation.The cause of POF is still unknown;however,autoimmunity,familial inheritance,metabolism,and environmental pollution have been associated with POF.It is of great significance to clarify the specific pathogenesis of POF to treat female birth defects.Protein prenylation is a common post-translational modification of proteins that occurs primarily at the cysteine residues of the protein’s carbon-terminal CaaX motif.It uses farnesyl pyrophosphate(FPP)or geranylgeranyl pyrophosphate(GGPP)as a lipid donor,which can increase the hydrophobicity of the protein and promote membrane association.Prenylation of small G proteins such as Ras is indispensable for their signal transduction function.Geranylgeranyl diphosphate synthase(GGPPS)is a branch-point enzyme in the mevalonate pathway.Our previous study found that GGPPS can participate in the development and differentiation of spermatogonia,and the transition of primary/secondary follicles through the regulation of prenylation of H-Ras,Rheb,Rho and other proteins.Therefore,we speculate that GGPPS may be involved in the regulation of POF.In this study,we examined the expression pattern of GGPPS during ovarian aging and found that GGPPS decreased with ovarian aging.The oxidative stress senescence theory inspired us to detect reactive oxygen species(ROS)in aging ovary and its effect on the expression of GGPPS.We found that the ROS level in the ovary of aged mice was significantly higher than that of young mice.At the cellular level,we treated the oocytes with hydrogen peroxide to mimic the aging process.The level of ROS in oocytes after treatment with hydrogen peroxide was significantly increased,and ROS did reduce the expression of GGPPS protein in oocytes without affecting mRNA levels.It was confirmed through MG132 rescue experiments that the regulation of ROS on GGPPS was mainly achieved through ubiquitination.Thus,our experiments show that accumulated ROS can trigger GGPPS ubiquitination degradation during ovarian aging.In order to study the role of GGPPS in the aging process of the ovary,we constructed an oocyte-specific Ggpps knockout mouse and found that compared with the control mice,the ovarian volume of knockout mice was significantly reduced,and the number of follicles and fertility were dramatically decreased,similar to aging mouse,Supplement of geranylgeraniol(GGOH),which can be converted to GGPP,significantly increased fertility in older female mice and restored mitochondrial function,spindle formation/attachment,and meiosis-related gene expression in the ovaries.It shows that GGOH can reverse ovary aging and restore fertility.Previous studies have shown that GGPPS-regulated protein prenylation participates in the glucose homeostasis of muscles and islets.Considering the destruction of ovarian function by metabolic syndrome,we hypothesize that the regulation of ovarian aging by GGPPS may be achieved through glucose metabolism.We found that the number of mitochondria in the ovary of older mice was significantly reduced,and the expression of key enzymes in glucose metabolism were increased,whereas the genes related with quality of oocytes were reduced.In order to further clarify the effect of glucose metabolism on ovarian aging,we gave mice a regular chow diet(RC),a high-fat diet(HFD),and a ketogenic diet(KD)for 5 months.The results of fertility experiments showed that the number of pups per litter in RC group gradually decreased over time,and the HFD group accelerated the process.The ketogenic diet did not contain carbohydrates such as glucose and showed the capacity of anti-ovarian aging.It is worth noting that the expression of the rate-limiting enzymes related to glycolysis in the ovary of Ggpps-knockout mice were elevated.Consistently,pyruvate and lactate were increased,whereas ATP was decreased.Above results show that oocyte-specific knockout of Ggpps elevates anaerobic glycolysis in the ovary.In summary,our results show that GGPPS is a key factor regulating POF.Accumulation of ROS during aging can degrade GGPPS protein through ubiquitination,promoting anaerobic glycolysis in the ovaries,and accelerate aging.In addition,our results suggest that exogenous supplementation of GGOH may be one of the methods for delaying ovarian aging and treating POF.