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.

Eukaryotic translation initiation factor 2 subunit 2 (EIF2S2), a subunit of the heterotrimeric G protein EIF2, is involved in the initiation of translation. Our findings demonstrate that the depletion of Eif2s2 in premeiotic germ cells causes oocyte arrest at the pachytene and early diplotene stages at 1 day postpartum (dpp) and 5 dpp, respectively, and eventually leads to oocyte apoptosis and failure of primordial follicle formation. Further studies reveal that Eif2s2 deletion downregulates homologous recombination‐related and mitochondrial fission‐related protein levels, and upregulates the integrated stress response‐related proteins and mRNA levels. Consistently, Eif2s2 deletion significantly decreases the expression of dictyate genes and compromises mitochondrial function, characterized by elongated shapes, decreased ATP levels and mtDNA copy number, along with an excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Furthermore, DNA damage response and proapoptotic protein levels increase, while anti‐apoptotic protein levels decrease in Eif2s2‐deleted mice. An increase in oocytes with positive cleaved‐Caspase‐3 and TUNEL signals, alongside reduced Lamin B1 intensity, further indicates oocyte apoptosis. Collectively, Eif2s2 deletion in premeiotic germ cells causes oocyte meiotic arrest at the early diplotene stage by impairing homologous recombination, and eventually leads to oocyte apoptosis mainly through the downregulation of mitochondrial fission‐related proteins, ROS accumulation and subsequent DNA damage.

Proto-oncogenic receptor tyrosine kinase (KIT) ligand (KITL) secreted by granulosa cells and its receptor KIT on oocytes are crucial for primordial follicle formation and activation, and follicular development. In the present study, ZnSO₄ decreased the number of primordial and growing follicles in cultured neonatal mouse ovaries when KITL-KIT signaling was inhibited by ISCK03. ZnSO₄ also significantly increased the mRNA and protein levels of Zrt/Irt-like protein 6 (ZIP6, a zinc importer) and zinc levels in the oocytes of cultured neonatal mouse ovaries in the presence of ISCK03, suggesting that the increase in ZIP6 levels results in zinc overload in the oocytes of cultured neonatal mouse ovaries. Further experiments indicated that zinc overload resulted in oocyte apoptosis in cultured neonatal mouse ovaries via oxidative stress-driven dual mechanisms: irreversible DNA damage in the nucleus and autophagic flux blockade in the cytoplasm of oocytes. Moreover, the intraperitoneal injection of ZnSO₄ and ISCK03 significantly increased ZIP6 expression, DNA damage, autophagic flux blockade, and apoptosis of oocytes in neonatal mice. Taken together, these findings indicate that granulosa cell-secreted KITL is involved in maintaining zinc homeostasis in the oocytes of neonatal mouse ovaries. This study not only reveals a novel function of granulosa cells in supporting oocyte homeostasis, but also provides a theoretical basis for identifying individuals susceptible to zinc dyshomeostasis caused by the impaired KITL-KIT signaling.

Chemotherapy causes primordial follicle apoptosis, resulting in premature ovarian insufficiency (POI) and infertility. In this study, we found that intraperitoneal injection of retinoic acid (RA) and calcitriol partially reversed the cyclophosphamide and doxorubicin treatment-induced decrease in primordial follicles in neonatal mouse ovaries. Furthermore, RA and calcitriol co-treatment reversed cyclophosphamide treatment-induced PI3K/Akt activity and FOXO3a nuclear export in the oocytes within primordial follicles, suggesting that the oocyte transcriptional activity was decreased, which in turn reduced the binding of chemotherapeutic drugs to DNA. Consistent with these findings, RA and calcitriol co-treatment reversed cyclophosphamide treatment-induced changes in reactive oxygen species (ROS), DNA damage response proteins (γH2AX, p-CHK2, p-p53, PUMA, BAX, Cleaved Caspase-3, and cPARP), and antioxidant proteins (NRF2, HO-1, and GPX4). Moreover, RA and calcitriol co-treatment preserved fertility in cyclophosphamide-treated mice without impairing cyclophosphamide’s antitumor efficacy in MCF-7 tumor-bearing mice. Thus, RA and calcitriol protect mouse primordial follicles from cyclophosphamide treatment-induced apoptosis by inhibiting cyclophosphamide treatment-induced oocyte transcriptional activity and enhancing antioxidant capacity. Our results suggest a potential strategy for preserving ovarian reserve during chemotherapy in female cancer patients.

Mutations in several translation initiation factors are closely associated with premature ovarian insufficiency (POI). In this study, we demonstrated that the conditional knockout of eukaryotic initiation factor 2 (eIF2) subunits Eif2s1 and Eif2s2 in mouse oocytes caused oocyte apoptosis within the early growing follicles. Subsequent research indicated that the depletion of Eif2s2 in oocytes reduced the levels of mitochondrial fission-related proteins (p-DRP1, FIS1 and MFF) and increased the mRNA and protein levels of the integrated stress response (ISR)-related factors (ASNS, SLC7A1, GRB10 and PSAT1). Consistent with this, the depletion of Eif2s2 in oocytes resulted in mitochondrial dysfunction characterized by elongated form, aggregated distribution beneath the oocyte membrane, decreased mitochondrial membrane potential and ATP content, and excessive accumulation of reactive oxygen species (ROS). At the same time, the depletion of Eif2s2 in oocytes led to increased levels of DNA damage response proteins (γH2AX, p-CHK2 and p53) and proapoptotic proteins (BAX and PARP1), as well as decreased the levels of anti-apoptotic protein BCL-xL. Collectively, these findings indicate that the depletion of eIF2 subunits in mouse oocytes leads to oocyte apoptosis within the early growing follicles, attributed to the impaired translation of mitochondrial dynamics regulatory proteins and then the upregulated ROS levels and DNA damage. This study provides new insights into pathogenesis and genetic diagnosis for POI.