Multi‐Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

Abstract 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.


Supplementary Figures:
Violin plots showing the TE changes in genes not enriched by m6A and m6A-enriched genes.p-Value was calculated with Student's t-test for independent samples.b) Violin plots showing the TE changes in group not enriched by m6A and m6A-enriched group genes in the YTHDF3-KD group oocytes compared with the control group oocytes.p-Values were calculated with one-way ANOVA and Bonferroni post-hoc test.c) Venn diagram showing the overlaps of the m6A-enriched gene set, high-TE genes in the control group oocytes, and high-TE genes in the YTHDF3-KD group oocytes.High TE, TE>2.d) Bar plots showing the numbers of high-TE genes (TE>2) and low-TE genes (TE<0.5)among the four groups of oocytes.Red denotes high-TE genes.Blue denotes low-TE genes.e) Violin plots showing the TE changes in genes of the papCPEcontaining group and genes of the group not containing papCPE in the YTHDF3-KD group oocytes compared with the control group oocytes.p-Value was calculated with Student's t-test for independent samples.f) Violin plots showing the TE changes in the genes of the CPE-containing group and the genes of the group not containing CPE in the YTHDF3-KD group oocytes compared with the control group oocytes.p-Value was calculated with Student's t-test for independent samples.g) Violin diagram depicting the change in TE upon YTHDF3 depletion among the four groups of genes.p-Values were calculated with one-way ANOVA and Bonferroni post-hoc test.h) Violin plots showing the TE changes in four groups of genes in the YTHDF3-KD group oocytes and the control group oocytes.p-Values were calculated with one-way ANOVA and Bonferroni post-hoc test.i) The distribution and enrichment of YTHDF3-binding sites within different gene regions.j) GO term enrichment analysis of the overlapping YTHDF3 target genes described in Figure .5c. k) Violin plots showing the TE changes in YTHDF3-target group and non-YTHDF3-target group genes in oocytes upon YTHDF3 depletion.p-Value was calculated with Student's t-test for independent samples.l) Overlapping analysis of human m6A-enriched genes (m6A-Atlas database), mouse m6A-containing genes (RMBase database), and YTHDF3 binding genes identified in HEK293T cells.YTHDF3-KD, YTHDF3 knockdown.TE, translational efficiency.CPEs, cytoplasmic polyadenylation elements.papCPE, CPEs residing near PAS, <100 nt.GO, Gene ontology.Ns, no significant difference.**P<0.01,***P<0.001,****P<0.0001.Red and blue dots denote up-and down-regulated genes, respectively.P<0.05, FC>1.5 or <0.67.e) Representative KEGG analysis of the overlapped translationally upregulated genes between aged mouse and human oocytes.f) Representative KEGG analysis of the overlapped translationally upregulated genes between aged mouse and human oocytes.PCA, principal component analysis.DEGs, differentially expressed genes.FC, fold change.KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure S1 .
Figure S1.Multi-omics analyses of aged and young mouse oocytes.a) The number of germinal vesicle (GV)-stage oocytes retrieved from young and aged mice.Each dot represents a single biological replicate.p-Values were calculated with Student's t-test for paired samples.b) The in vitro PB1 emission rates of oocytes from young and aged

Figure S2 .
Figure S2.Translational efficiency of aged and young mouse oocytes.a) Volcano plot showing the changes in the RNA decay-related genes identified by translatomics.b) Volcano plot showing the changes in the histone modification regulatory genes identified by the translatomics.c) Gene set enrichment analysis of TE showing the downregulated genes enriched in the hallmark of the G2/M checkpoint and the upregulated genes enriched in the hallmark of oxidative phosphorylation.d) Gene ontology term enrichment of the young-specific high-TE genes of aged mouse oocytes.TE, translational efficiency.

Figure S3 .
Figure S3.Correlations of translational efficiency changes and m6A modification/CPE existence in aged mouse oocytes.a) Bar plots showing the numbers of up-and downregulated TE genes between aged and young mouse oocytes.Pink denotes the m6Aenriched genes.Blue denotes genes not containing m6A. b) Violin plots showing the changes in CPE-containing and non-CPE-containing genes in aged mouse oocytes.CPE, cytoplasmic polyadenylation element.TE, translational efficiency.p-Value was calculated with Student's t-test for independent samples.Ns, no significant difference.

Figure S4 .
Figure S4.Immunofluorescence verifying the expression of YTHDF3 in young mouse

Figure S5 .
Figure S5.The translational and transcriptional landscapes of oocytes upon YTHDF3 depletion.a) Heatmap depicting the Pearson correlation coefficient of the translatomics between each biological replicate from the control group oocytes and the YTHDF3-KD group oocytes.b) Heatmap depicting the Pearson correlation coefficient of the transcriptome between each biological replicate from the control group oocytes and the YTHDF3-KD group oocytes.c) PCA plot of the translatomics in oocytes from the control group and the YTHDF3-KD group.d) PCA plot of the transcriptomics in oocytes from the control group and the YTHDF3-KD group.e,f) Volcano diagram showing DEGs detected by the single-cell translatomics (e) and transcriptomics (f).Red

Figure S7 .
Figure S7.The RNA levels of HELLS genes identified by RT-qPCR in HEK293T cells.a) Scatter plot showing the correlation in gene translational log2 FC between YTHDF3-KD/control oocytes and aged/young oocytes.The Pearson correlation coefficient=0.2, with a p-Value < 0.001.b) Shown are m6A RIP followed by qPCR with 50 mouse oocytes on different sites of the Hells gene.UTR3-1, primers at the 3'UTR of Hells, CDS-1 and CDS-2, two pairs of primers at the CDS region of Hells.p-Value was calculated with two tailed Mann-Whitney test.c) M6A RIP followed by RT-qPCR in HEK293T cells confirming the enrichment of m6A in HELLS mRNA.p-Value was calculated with two tailed Mann-Whitney test.d) Distribution of the YTHDF3-binding site across the HELLS gene in HEK293T cells.e) YTHDF3 RIP followed by RT-qPCR in HEK293T cells confirming the interaction between YTHDF3 and HELLS mRNA.p-Value was calculated with two tailed Mann-Whitney test.f) The RNA levels of the HELLS gene in HEK293T cells transfected with empty vector or wild-type (YTHDF3-WT) or mutant (YTHDF3-Mut) Flag-tagged YTHDF3 plasmid.p-Value was calculated with two tailed Mann-Whitney test.FC, fold change.RIP, RNA immunoprecipitation.NC, negative control.Ns, no significant difference.**P<0.01.

Figure S8 .
Figure S8.The transcriptional and translational landscapes of aged human oocytes.a) Heatmap depicting the Pearson correlation coefficient of the translatomics between each biological replicate from the young human oocytes and the aged human oocytes.b) Heatmap depicting the Pearson correlation coefficient of the transcriptomics between each biological replicate from the young human oocytes and the aged human oocytes.c) PCA plot of the transcriptomics of oocytes from young and aged human females.d) Volcano diagram showing DEGs detected by ultrasensitive transcriptomics.Red and blue dots denote up-and down-regulated genes, respectively.P<0.05, FC>1.5 or <0.67.e) Representative KEGG analysis of the overlapped translationally upregulated genes between aged mouse and human oocytes.f) Representative KEGG analysis of the overlapped translationally upregulated genes between aged mouse and human oocytes.PCA, principal component analysis.DEGs, differentially expressed genes.FC, fold change.KEGG, Kyoto Encyclopedia of Genes and Genomes.

Table S2 .
The changes of 302 identified genes in translational efficiency (TE) and translatomics

Table S3 .
RNA-binding proteins that were differently expressed between aged and young human oocytes