(301) N6-methyladenosine RNA methylation-related gene expression during preantral-to-antral follicle transition

Fuhua Xu¹, Jing Xu², Hau Li¹, Peixin Yang¹

1. Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, USA
2. Department of Biology & Chemistry, Liberty University, Lynchburg, USA

Abstract Text: One of the main functions of the ovary is to support follicle growth and maturation. The process of preantral-to-antral follicle transition involves a large scale of gene expression alterations, including not only RNA transcription but also post-transcriptional regulation. N6-methyladenosine (m6A) RNA methylation is the most prevalent internal modification of messenger RNA (mRNA) and non-coding RNA. The m6A modification precisely regulates gene expression and RNA metabolism, including pre-mRNA processing, as well as RNA transport, localization, splicing, stability, degradation, and translation. Therefore, m6A plays an important role in embryonic development, tumor occurrence, organ development, and other physiological or pathological processes involving post-transcriptional regulations. Recent studies discovered that methyltransferase-like 3 (Mettl3) and Mettl14 are key factors in RNA modification, specifically in the methylation of adenosine residues to form m6A, which are termed as m6A writers. The dynamic regulation of m6A modification on RNA also requires fat mass and obesity-associated protein (Fto) and AlkB homolog 5 (Alkbh5), so called m6A erasers, which function in RNA metabolism by removing m6A from RNA. In addition, YT521-B homology m6A RNA binding protein 1 (Ythdf1) and insulin-like growth factor 2 mRNA binding protein 3 (Igf2bp3) are the key m6A reader proteins. Ythdf1 and Igf2bp3 recognize and bind to m6A-modified mRNA, influencing various aspects of RNA metabolism, including mRNA stability, translation, and decay. To understand the potential involvement of m6A during preantral-to-antral follicle transition, an exploratory study was designed to investigate differences in the expression of m6A writers, erasers, and readers between preantral and antral follicles. Ovaries were collected from adult female CD-1 mice (8 weeks old; n = 3). Follicles were isolated mechanically, including preantral follicles with multiple granulosa cell layers ( >150 μm in diameter) and antral follicles (< 300 μm in diameter), and pooled (20 preantral or 10 antral follicles) for each animal. Total RNA was extracted using an Absolutely RNA Nanoprep Kit for reverse transcription. Quantitative real-time PCR was employed to determine the gene expression levels of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Igf2bp3 using TaqMan Gene Expression Assays. Mitochondrial ribosomal protein S10 (Mrps10) served as internal control. Statistical analysis was performed using a Student’s t-test. The mRNA levels of Mettl3, but not Mettl14, in antral follicles were lower than those of preantral follicles (1.093 ± 0.019 versus 1.251 ± 0.018; P = 0.026). The mRNA levels of Fto and Alkbh5 were comparable between preantral and antral follicles. In addition, the mRNA levels of Ythdf1, but not Igf2bp3, decreased after the antrum formation (1.062 ± 0.009 versus 0.9748 ± 0.010; P = 0.021). These results suggest that m6A-associated RNA methylation has a potential regulatory effect on RNA turnover during preantral-to-antral follicle transition. The decreased expression of m6A writer and reader results in increased degradation of RNA molecules essential for preantral follicle growth which are replaced by those critical for the subsequent antrum formation and antral follicle maturation.