In mammals, nonrenewable primordial follicles are activated in an orderly manner to maintain the longevity of reproductive life. Mammalian target of rapamycin (mTOR)-KIT ligand (KITL) signaling in pre-granulosa cells and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-forkhead Box O3a (FOXO3a) signaling in oocytes are important for primordial follicle activation. The activation process is accompanied by the enhancement of energy metabolism, but the causal relationship is unclear. In the present study, the levels of glycolysis-related proteins GLUT4, HK1, PFKL, and PKM2 were significantly increased in granulosa cells but were decreased in oocytes during the mouse primordial-to-primary follicle transition. Both short-term pyruvate deprivation in vitro and acute fasting in vivo increased the glycolysis-related gene and protein levels, decreased AMPK activity, and increased mTOR activity in mouse ovaries. The downstream pathways Akt and FOXO3a were phosphorylated, resulting in mouse primordial follicle activation. The blockade of glycolysis by 2-deoxyglucose (2-DG), but not the blockade of the communication network between pre-granulosa cells and oocyte by KIT inhibitor ISCK03, decreased short-term pyruvate deprivation-promoted mTOR activity. Glycolysis was also increased in human granulosa cells during the primordial-to-primary follicle transition, and short-term pyruvate deprivation promoted the activation of human primordial follicles by increasing the glycolysis-related protein levels and mTOR activity in ovarian tissues. Taken together, the enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling. These findings provide new insight into the relationship between glycolytic disorders and POI/PCOS.

Germ cell division and differentiation require intimate contact and interaction with the surrounding somatic cells. Luteinizing hormone (LH) triggers epidermal growth factor (EGF)-like growth factors to promote oocyte maturation and developmental competence by activating EGF receptor (EGFR) in somatic cells. Here, we showed that LH-EGFR signaling-activated sphingosine kinases (SphK) in somatic cells. The activation of EGFR by EGF increased S1P and calcium levels in cumulus-oocyte complexes (COCs), and decreased the binding affinity of natriuretic peptide receptor 2 (NPR2) for natriuretic peptide type C (NPPC) to release the cGMP-mediated meiotic arrest. These functions of EGF were blocked by the SphK inhibitor SKI-II, which could be reversed by the addition of S1P. S1P also activated the Akt/mTOR cascade reaction in oocytes and promoted targeting protein for Xklp2 (TPX2) accumulation and oocyte developmental competence. Specifically depleting Sphk1/2 in somatic cells reduced S1P levels and impaired oocyte meiotic maturation and developmental competence, resulting in complete female infertility. Collectively, SphK-produced S1P in somatic cells serves as a functional transmitter of LH-EGFR signaling from somatic cells to oocytes: acting on somatic cells to induce oocyte meiotic maturation, and acting on oocytes to improve oocyte developmental competence.

Background: Aged women and premature ovarian insufficiency (POI) patients have residual dormant primordial follicles that are hard to be activated through a physiological process. However, there are no effective and safe drugs to help them. Methods: We used the in vitro culture model of newborn mouse ovaries to identify the drugs that promote primordial follicle activation and study its mechanisms. It was verified by in vivo injection model of newborn mice and in vitro culture model of human ovarian tissue. In addition, we used the aged mice as a low infertility model to verify the effects of primordial follicle activation, and fertility by drugs. Results: Eleven metallic compounds activated mouse primordial follicles, and the five most effective compounds were selected for further study. Thapsigargin (TG), CrCl 3 , MnCl 2 , FeCl 3 and ZnSO 4 increased the levels of the glycolysis-related proteins (glucose transporter type 4, GLUT4; hexokinase 1, HK1; pyruvate kinase M2, PKM2; phosphofructokinase, liver type, PFKL), phosphorylated mammalian target of rapamycin (p-mTOR) in cultured mouse ovaries. The compound-promoted p-mTOR levels could be completely blocked by 2-DG (the inhibitor of glycolysis). The compounds also increased the levels of phosphorylated protein kinase B (p-Akt). TG-, CrCl 3 - and FeCl 3 -promoted p-Akt levels, but not MnCl 2 - and ZnSO 4 - promoted p-Akt levels, could be completely blocked by ISCK03 (the inhibitor of proto-oncogenic receptor tyrosine kinase, KIT). The injection of newborn mice with the compounds also activated primordial follicles and increased the levels of the glycolysis-related proteins, p-mTOR, and p-Akt. The oral administration of the compounds in adolescent and aged mice promoted primordial follicle activation, and had no obvious side effect. Importantly, ZnSO 4 also increased ovulated oocytes, oocyte quality and offspring in aged mice. Furthermore, the compounds promoted human primordial follicle activation and increased the levels of the glycolysis-related proteins, p-mTOR, and p-Akt. Conclusion: The metallic compounds activate primordial follicles through the glycolysis-dependent mTOR pathway and/or the PI3K/Akt pathway, and the oral administration of ZnSO 4 enhances fertility in aged mice. We suggest that these metallic compounds may be oral drugs to ameliorate fertility deficits in aged women and POI patients.

Germ cells division and differentiation requires intimate contact and interaction with the surrounding somatic cells. Luteinizing hormone (LH) triggers epidermal growth factor (EGF)-like growth factors to promote oocyte maturation and developmental competence by activating EGF receptor (EGFR) in somatic cells. Here, we show that LH-EGFR signaling activates sphingosine kinases (SphK1 and SphK2) of somatic cells to generate sphingosine-1-phosphate (S1P). S1P increases phospholamban (PLN) and calcium levels of cumulus cells and then decreases the binding affinity of natriuretic peptide receptor 2 (NPR2) for natriuretic peptide type C (NPPC), thus releasing the cGMP-mediated meiotic arrest. S1P also activates the Akt/mTOR cascade reaction in oocytes to promote targeting protein for Xklp2 (TPX2) accumulation and oocyte developmental competence. Specifically depleting Sphk1/2 in somatic cells reduces S1P levels and impairs oocyte meiotic maturation and developmental competence, resulting in complete female infertility. Collectively, SphK-produced S1P in somatic cells serves as a functional transmitter of LH-EGFR signaling from somatic cells to oocytes: acting on somatic cells to induce oocyte meiotic maturation, and acting on oocytes to improve oocyte developmental competence.