NAT10 Maintains OGA mRNA Stability Through ac4C Modification in Regulating Oocyte Maturation

Abstract

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.

Keywords: N4-acetylcytidine; NAT10; O-GlcNAc; OGA; in vitro maturation; oocyte; transcription.

Figure 1

Expression profiling of NAT10-depleted mouse oocytes. (A)Volcano map showed the gene expression with NAT10 KD. Blue dots represented downregulation, and red dots represented upregulation. (B) The heatmap showed clusters of differential expression of genes. (C) Pie chart presented the proportion of up/downregulated genes in 280 DEGs. (D) Cellular amino acid metabolic process and cytoplasmic sequestering of protein were main biological process of 280 DEGs. (E) A total of 280 DEGs were enriched in metabolism and signal transduction according to KEGG enrichment analysis.

Figure 2

Transcripts modulated by NAT10-mediated ac4C modification in oocytes. (A) Venn diagram and pie chart showed the proportion of NAT10-regulated ac4C genes and non–ac4C-modified genes. (B) Box diagram displayed the fold change difference between NAT10-regulated ac4C-modified DEGs and non–ac4C-modified DEGs. (C) Sixteen downregulated ac4C-modified DEGs regulated by NAT10 mainly participated in regulation of protein binding and positive regulation of transcription, DNA-templated. (D) KEGG analysis showed 16 downregulated ac4C-modified DEGs regulated by NAT10 were enriched in cancer, signal transduction, folding, sorting, and degradation. Unpaired t-test was used in (B) to compare the expression of genes because the number of genes did not match between two groups. **P < 0.01.

Figure 3

OGA Stability was modulated by NAT10-mediated ac4C modification. (A) Expression of OGA was downregulated significantly by comparing NAT10 KD transcriptome with the control. (B) The diagram displayed ac4C-modified potential sites of OGA in homo sapiens and Mus musculus. (C) Bar chart showed OGA could bind to NAT10 and be ac4C-modified by RIP. (D) The degradation of OGA was suppressed in NAT10-overexpressing compared with negative control group. (E) Immunofluorescence showed NAT10 degradation and OGA downregulation after NAT10 KD by Trim-Away. Data represent the mean ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P< 0.001.

Figure 4

OGA depletion retarded mouse oocyte maturation in vitro. (A) Compared with GV oocytes, OGA gene expression was upregulated in MII oocytes. (B) OGA was significantly downregulated by Trim-Away. (C) Box plot showed the difference of oocyte maturation rates between OGA KD group and the control group. (D) Representative pictures of oocyte maturation in OGA KD group and the control group. (E) Different expression of O-GlcNAc in GV and MII oocytes was verified by immunofluorescence, and O-GlcNAc modification was upregulated after OGA intervention. Data represent the mean ± SEM of at least three independent experiments. *P < 0.05.

Figure 5

Expression Profiling of Oocytes with OGA Knockdown. (A) Volcano map showed global genes change after OGA KD, with blue dots representing downregulation and red dots representing upregulation. (B) Heatmap showed the overall clusters of differential expression of genes. (C) A total of 3,514 DEGs with OGA KD were mainly associated with metabolic processes according to GO enrichment. (D) KEGG analysis displayed top 20 pathways where 3,514 DEGs were involved in. (E) Pie chart showed the distribution of DEGs by joint analysis of OGA KD, NAT10-KD transcriptomes, and ac4C RIP data.

Figure 6

Joint Analysis of NAT10-depleted and OGA-depleted Transcriptomes. (A–C) GSEA analysis showed that NAT10 KD transcriptome genes were mainly enriched in G protein–coupled receptor activity, molecular transducer activity, and nucleosomal DNA binding. (D–F) OGA-depleted expression profiling was enriched in similar gene sets as NAT10-KD according to GSEA analysis.

Figure 7

Co-regulated genes of NAT10 and OGA in oocyte maturation. (A) Common upregulated DEGs and their expression in NAT10 KD and OGA KD transcriptomes. (B) Common downregulated DEGs and expression in NAT10 KD and OGA KD transcriptomes. *P < 0.05 and **P < 0.01.