Meijia Zhang

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 translation initiation factor 2B (eIF2B) subunits Eif2b2, Eif2b3, Eif2b4 or Eif2b5 , but not Eif2b1 , in mouse oocytes of primordial follicles caused oocyte apoptosis within the early growing follicles. Integrative Ribo-seq and RNA-seq analysis revealed the upregulation of integrated stress response- and DNA damage-related genes, and the downregulation of oocyte development-related genes in Eif2b4 -cKO oocytes. Then, further research indicated that the depletion of Eif2b4 in oocytes resulted in mitochondrial dysfunctions characterized by decreased mitochondrial membrane potential, mtDNA copy numbers, and ATP content, along with excessive accumulation of reactive oxygen species (ROS). At the same time, the depletion of Eif2b4 in oocytes resulted in the increased levels of DNA damage response (DDR) proteins (NBS1, p-ATM, p-CHK2, and p53) and proapoptotic protein BAX, as well as the decreased levels of DNA damage repair protein Polβ and anti-apoptotic protein BCL-xL. Collectively, these findings indicate that the depletion of eIF2B subunits in mouse oocytes leads to oocyte apoptosis within the early growing follicles by DNA damage and mitochondrial dysfunction. This study provides new insights into the pathogenesis and genetic diagnosis of POI.

Premature ovarian insufficiency (POI) patients usually have a few residual dormant primordial follicles that are difficult to activate. We investigated the effects of branched‐chain amino acids (BCAAs) on mouse primordial follicle activation. Leucine, but not isoleucine and valine, promoted primordial follicle activation in the cultured neonatal mouse ovaries. Furthermore, leucine significantly elevated the levels of phosphorylated protein kinase B (p‐Akt) and phosphorylated forkhead Box O3a (p‐FOXO3a), and increased the number of oocytes exhibiting nuclear export of FOXO3a in mouse ovaries. LY294002 and PI3K‐IN‐1, the inhibitors of the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway, reversed the promoting effects of leucine on mouse primordial follicle activation. Importantly, intraperitoneal injection of leucine in 3‐day‐old neonatal mice and oral administration of leucine in 21‐day‐old mice with or without cyclophosphamide treatment significantly increased the number of growing follicles. Thus, leucine activates mouse primordial follicles through the PI3K/Akt pathway. Leucine may be a potential oral supplement for treating infertility in POI patients with residual dormant primordial follicles.

In mammals, precise regulation of primordial follicle activation is essential for maintaining the female reproductive lifespan. In this study, we demonstrated that the vitamin D receptor (VDR) is expressed in both pre-granulosa cells and oocytes within primordial follicles of mice and humans. The VDR ligand, calcitriol, was tested through ovarian culture and intraperitoneal injection experiments in neonatal mice. In addition, oral administration of the calcitriol precursor VD3 was performed in mice. Calcitriol was also tested on cultured human ovarian fragments. Calcitriol inhibited the activation of primordial follicles in neonatal mice. Moreover, calcitriol promoted the formation of the VDR-RXR-p85α complex, thereby stabilizing the PI3K heterodimer (p85α/p110) and inhibiting activation of the PI3K/Akt signaling pathway. This resulted in reduced phosphorylation of FOXO3a in the ovaries and decreased nuclear export in oocytes of primordial follicles, thereby suppressing the activation of primordial follicles. Oral administration of VD3 significantly decreased the number of growing follicles and prolonged the reproductive age in mice compared with controls. Importantly, calcitriol also inhibited human primordial follicle activation and reduced p-Akt levels in cultured ovarian fragments. Thus, calcitriol inhibits the activation of both mouse and human primordial follicles by suppressing the VDR-mediated PI3K/Akt signaling pathway. As an oral medication, the calcitriol precursor VD3 may be used to maintain the primordial follicle reserve and extend the reproductive age in women.

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.

In each wave, only a small portion of primordial follicles are selectively activated into the growing follicular pool, and the majority of primordial follicles remain in a relatively quiescent state. Hypoxanthine is present in the follicular fluid with high concentrations and inhibits phosphodiesterase activity. In this study, hypoxanthine synthase purine nucleoside phosphorylase (Pnp) was expressed in oocytes, and its expression levels increased progressively with oocyte growth, accompanied by an increase in ovarian hypoxanthine levels. Ovarian hypoxanthine levels were significantly increased in equine chorionic gonadotropin-primed mice, accompanied by an increase in primary follicle numbers. Hypoxanthine significantly increased the number of growing follicles and the levels of cyclic adenosine monophosphate (cAMP) and phosphorylated protein kinase B (p-Akt) in cultured ovaries. Hypoxanthine with intraperitoneal injection in neonatal mice also significantly increased growing follicle numbers and p-Akt protein levels in the ovaries. Additionally, oral BCX-1777 in adolescent mice, a PNP inhibitor, significantly decreased hypoxanthine levels and growing follicle numbers in the ovaries. Thus, hypoxanthine promotes mouse primordial follicle activation through cAMP-PI3K/Akt pathway. The high levels of hypoxanthine produced by the preovulatory fully-grown oocytes may contribute to the promotion of the cyclic activation of primordial follicles.

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.

Idiopathic non-obstructive azoospermia (iNOA) is associated with reduced expression of multiple eukaryotic translation initiation factors ( EIFs) in spermatogonia, including eukaryotic translation initiation factor 5 ( EIF5). The present study revealed that eIF5 mRNA and protein levels were markedly higher in male mouse ( Mus musculus) germ cells than in Leydig or Sertoli cells. Thus, to define the role of eIF5 in spermatogenesis, Eif5 conditional knockout (cKO) mice were generated by crossing Eif5 ^fl/fl mice with Stimulated by retinoic acid 8 ( Stra8)-Cre mice. Loss of Eif5 in male germ cells reduced both SRY-box transcription factor 3 (SOX3) ⁺ progenitor spermatogonia and Kit proto-oncogene receptor tyrosine kinase (KIT) ⁺ differentiating spermatogonia, leading to severe meiotic failure and complete male infertility. Mechanistically, Eif5 deficiency impaired translation of genes involved in ubiquitination, autophagy, and DNA repair, which, in turn, triggered excessive endoplasmic reticulum (ER) stress and compromised DNA repair in SOX3 ⁺ progenitor spermatogonia. These defects promoted DNA damage and subsequent apoptosis, resulting in progressive germ cell depletion and sterility. Collectively, these findings highlight the essential role of eIF5 in spermatogenesis and offer a potential therapeutic strategy for iNOA associated with reduced expression of EIF5 and other translation initiation factors.

Vitamins, as essential trace organic compounds for the human body, are crucial substances in the maintenance of normal reproductive function. Nevertheless, the effects of vitamins on the activation of primordial follicles and the underlying mechanisms remain unclear. We used the in vitro culture model of newborn mouse ovaries to identify the vitamins that promote primordial follicle activation and study the mechanism of vitamin B6. The mechanism of VB6 was verified by in vivo injection model of newborn mice, oral feeding of adolescent mice, and in vitro culture model of human ovarian tissue. Thiamine monochloride (VB1), riboflavin (VB2), nicotinic acid (VB3), D-pantothenic acid (VB5), pyridoxine (VB6), folic acid (VB9), vitamin B12, L-ascorbic acid (VC), and tocopherol (VE) increased the number of growing follicles in the cultured newborn mouse ovaries. Next, we studied the molecular mechanism by using VB6. VB6 increased the protein levels of phosphorylated protein kinase B (p-Akt) and forkhead box O3a (p-FOXO3a), as well as the proportion of primordial follicle oocytes with FOXO3a nuclear export in the cultured mouse ovaries. The addition of PI3K inhibitor LY294002 blocked VB6-induced increase of growing follicles. Furthermore, intraperitoneal injection of VB6 in newborn mice and oral administration of VB6 to adolescent mice significantly increased the number of growing follicles and the protein levels of p-Akt. Importantly, VB6 also significantly increased the number of growing follicles and the protein levels of p-Akt in the cultured human ovarian tissues. Our findings indicate that VB6 promotes primordial follicle activation through the PI3K/Akt signaling pathway.