Abstract
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.
Highlights
In vitro maturation (IVM) of oocytes is an essential technique in clinical practice of assisted reproductive technology and research of reproductive biology (Walls et al, 2015; Saenz-de-Juano et al, 2019)
Immunoblot assays demonstrated that Transducin beta-like protein 3 (TBL3) was expressed in mouse oocytes, but no significant difference in expression was observed between germinal vesicle (GV) and metaphase II (MII) oocytes (Figure 5J)
With oocyte meiosis resuming triggered by hormones, approximately 20% of total maternal transcriptome were selectively degraded (Ivanova et al, 2017), while a large number of specific transcripts were translationally activated by MAPK pathway (Brachova et al, 2021). messenger RNA (mRNA) decay and translational regulation are fundamental events in oocyte meiotic maturation
Summary
In vitro maturation (IVM) of oocytes is an essential technique in clinical practice of assisted reproductive technology and research of reproductive biology (Walls et al, 2015; Saenz-de-Juano et al, 2019). The quality and developmental competence of oocytes maturated in vitro are still suboptimal (Walls et al, 2015; Li et al, 2021). Further investigation on the regulatory mechanisms of oocyte maturation is necessary. Transcription and translation are uncoupled during different stages of oocyte maturation. GV-stage oocytes complete meiosis I (MI) and advance to metaphase II (MII), accompanied by utilization of the assembled maternal mRNA. Oocyte maturation triggers a transition from mRNA stability to instability, leading to actively degradation of approximately 20% of total maternal transcriptome (Su et al, 2007; Ma et al, 2015). Post-transcriptional regulation underpinning mRNA stability and translation is a key determinant of gene expression during oocyte maturation (Ivanova et al, 2017). The detailed mechanism underlying the subtle regulation remains to be unraveled
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