Abnormal resumption of meiosis and decreased oocyte quality are hallmarks of maternal aging. Transcriptional silencing makes translational control an urgent task during meiosis resumption in maternal aging. However, insights into aging‐related translational characteristics and underlying mechanisms are limited. Here, using multi‐omics analysis of oocytes, it is found that translatomics during aging is related to changes in the proteome and reveals decreased translational efficiency with aging phenotypes in mouse oocytes. Translational efficiency decrease is associated with the N6‐methyladenosine (m6A) modification of transcripts. It is further clarified that m6A reader YTHDF3 is significantly decreased in aged oocytes, inhibiting oocyte meiotic maturation. YTHDF3 intervention perturbs the translatome of oocytes and suppress the translational efficiency of aging‐associated maternal factors, such as Hells, to affect the oocyte maturation. Moreover, the translational landscape is profiled in human oocyte aging, and the similar translational changes of epigenetic modifications regulators between human and mice oocyte aging are observed. In particular, due to the translational silence of YTHDF3 in human oocytes, translation activity is not associated with m6A modification, but alternative splicing factor SRSF6. Together, the findings profile the specific translational landscapes during oocyte aging in mice and humans, and uncover non‐conservative regulators on translation control in meiosis resumption and maternal aging.

In vitro maturation (IVM) refers to the process of developing immature oocytes into the mature in vitro under the microenvironment analogous to follicle fluid. It is an important technique for patients with polycystic ovary syndrome and, especially, those young patients with the need of fertility preservation. However, as the mechanisms of oocyte maturation have not been fully understood yet, the cultivation efficiency of IVM is not satisfactory. It was confirmed in our previous study that oocyte maturation was impaired after N-acetyltransferase 10 (NAT10) knockdown (KD). In the present study, we further explored the transcriptome alteration of NAT10-depleted oocytes and found that O-GlcNAcase(OGA) was an important target gene for NAT10-mediated ac4C modification in oocyte maturation. NAT10 might regulate OGA stability and expression by suppressing its degradation. To find out whether the influence of NAT10-mediated ac4C on oocyte maturation was mediated by OGA, we further explored the role of OGA in IVM. After knocking down OGA of oocytes, oocyte maturation was inhibited. In addition, as oocytes matured, OGA expression increased and, conversely, O-linked N-acetylglucosamine (O-GlcNAc) level decreased. On the basis of NAT10 KD transcriptome and OGA KD transcriptome data, NAT10-mediated ac4C modification of OGA might play a role through G protein–coupled receptors, molecular transduction, nucleosome DNA binding, and other mechanisms in oocyte maturation. Rsph6a, Gm7788, Gm41780, Trpc7, Gm29036, and Gm47144 were potential downstream genes. In conclusion, NAT10 maintained the stability of OGA transcript by ac4C modification on it, thus positively regulating IVM. Moreover, our study revealed the regulation mechanisms of oocytes maturation and provided reference for improving IVM outcomes. At the same time, the interaction between mRNA ac4C modification and protein O-GlcNAc modification was found for the first time, which enriched the regulation network of oocyte maturation.

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Background: In-vitro-grow (IVG) of preantral follicles is essential for female fertility preservation, while practical approach for improvement is far from being explored. Studies have indicated that neurotrophin-4 (NT-4) is preferentially expressed in human preantral follicles and may be crucial to preantral follicle growth.Methods: We observed the location and expression of Tropomyosin-related kinase B (TRKB) in human and mouse ovaries with immunofluorescence and Western blot, and the relation between oocyte maturation and NT-4 level in follicular fluid (FF). Mice model was applied to investigate the effect of NT-4 on preantral follicle IVG. Single-cell RNA sequencing of oocyte combined with cell-specific network analysis was conducted to uncover the underlying mechanism of effect.Results: We reported the dynamic location of TRKB in human and mouse ovaries, and a positive relationship between human oocyte maturation and NT-4 level in FF. Improving effect of NT-4 was observed on mice preantral follicle IVG, including follicle development and oocyte maturation. Transcriptome analysis showed that the reparative effect of NT-4 on oocyte maturation might be mediated by regulation of PI3K-Akt signaling and subsequent organization of F-actin. Suppression of advanced stimulated complement system in granulosa cells might contribute to the improvement. Cell-specific network analysis revealed NT-4 may recover the inflammation damage induced by abnormal lipid metabolism in IVG.Conclusions: Our data suggest that NT-4 is involved in ovarian physiology and may improve the efficiency of preantral follicle IVG for fertility preservation.

Patients with systemic lupus erythematosus (SLE) often have decreased fertility. Gene translation is crucial to oocyte meiosis and development. However, it remains unclear how SLE affects this process. Here, we used single-cell transcriptome and translatome sequencing to uncover a notable disruption in protein translation in oocytes from SLE mice, associated with the N ⁴ -acetylcytidine (ac ⁴ C) modification. Inhibition of ac ⁴ C levels in vitro substantially reduced oocyte translation efficiency. Notably, through trace-cell ac ⁴ C-RNA immunoprecipitation (acRIP) sequencing, we mapped the ac ⁴ C landscape in SLE mouse oocytes and found that deficient ac ⁴ C modification substantially impaired the translation of Zygote arrest 1 . Furthermore, we demonstrated that nicotinamide treatment notably and safely improved the quantity and quality of oocytes in SLE mice by enhancing ac ⁴ C modification levels. Our findings highlight the essential role of N - acetyltransferase 10 (NAT10)-mediated ac ⁴ C modification in abnormal oocyte development in SLE and suggest that nicotinamide holds promise for improving fertility in patients with SLE.