The Role of N6-methyladenosine Modification in Gametogenesis and Embryogenesis: Impact on Fertility.

Two gametes combine after fertilization to form a new individual. In human embryos, gene expression begins at the one-cell stage [ 109 ]. During transcriptional silencing following fertilization, the genome is reprogramed to allow the embryo to develop into a new individual [ 110 ]. The two primary windows of epigenetic reprogramming have been demonstrated in mouse models: (1) gametes are formed when oocytes undergo DNA demethylation, and thereafter, the genome is gradually remethylated, including imprinted genes and transposon regions; and (2) after fertilization, during early embryonic development, global demethylation (except for imprinted genes) and remethylation occur to establish a genealogy [ 111 , 112 ]. Through the MZT process, in which the breakdown of maternal products is synchronized with zygotic genome activation (ZGA), transcriptional control is transferred to the zygote [ 113 ]. An essential MZT event in animal embryos is the excision of a subset of maternal transcripts that accumulate during oogenesis. Invertebrates and vertebrates both have a maternally encoded mRNA decay mechanism (M-decay) that is activated before ZGA, while a second pathway that requires zygotic transcription clears more mRNAs afterward (Z-decay) [ 114 ]. In Z-decay and ZGA, the RNA expression of m 6 A transcripts (transposable elements MTA and MERVL) was greater than that of their unmarked counterparts, while M-decay transcripts did not significantly differ in the m 6 A status. Most maternal RNAs degrade during oocyte maturation and fertilization during the MZT process, and the initial wave of ZGA occurs between the late one-cell and late two-cell stages [ 96 ]. m 6 A methylation is essential for maintaining embryonic stem cells (ESCs) in a ground state [ 85 ]. Recent research has suggested that thousands of mouse ESC (mESC)-specific transcripts, including long intergenic non-coding RNAs, are changed by m 6 A and this alteration can control the fate of mESCs [ 115 ]. m 6 A-mediated maternal mRNA clearance is regulated by a zygotic program [ 13 ]. The overall abundance of m 6 A continuously decreases during MZT, and the number of m 6 A transcripts gradually increases after fertilization [ 96 ] ( Figure 4 ). The RNA-m 6 A modification landscape of human fetal tissues, however, reveals that several m 6 A peaks are present in introns and intergenic regions and that m 6 A is positively correlated with gene expression homeostasis, most likely by limiting or buffering gene expression perturbations at the post-transcriptional level [ 116 ]. During these periods of epigenetic recombination, genomic expression is highly affected by environmentally induced epigenetic defects. The m 6 A modification dynamically changes during embryonic preimplantation in mice, and it is higher in the blastocyst stage than in the two-cell, four-cell, and eight-cell stages [ 117 , 118 ]. Enzyme-linked immunosorbent assay showed significantly increased levels of m 6 A-marked RNAs in female germ cells (FGCs) at embryonic day 13.5 (E13.5) and E14.5 when compared with those at E12.5 [ 60 ]. The m 6 A content is markedly enriched in early embryogenesis in Drosophila but declines sharply 2 h after fertilization and stays low for the remainder of embryogenesis and the early larval stages [ 24 ]. However, in pigs, m 6 A methylation continues from the zygotic stage to the blastocyst stage and suddenly increases significantly during the transition from the morula to the blastocyst [ 82 , 119 ]. In summary, m 6 A undergoes dynamic changes during embryonic development. Changes in m 6 A and its regulators affect the progression of embryogenesis. Methyltransferases are highly conserved throughout evolution and are crucial for embryonic development [ 120 ]. Mettl3 KD impacts the decay of m 6 A ZGA transcripts [ 96 ]. In Mettl3 KO mice, nearly half of the two-cell stage embryos did not develop normally into four-cell stage cells, which impeded the MZT and ZGA processes [ 79 ]. KD of Mettl3 and Mettl14 in mESCs reduces m 6 A RNA methylation and results in a loss of self-renewal capacity [ 85 ]. Mettl14 KO mice, to a large extent, showed growth retardation and morphological abnormalities and died at E8.5 [ 121 ]. KIAA1429 is a newly discovered writer, and m 6 A generated via KIAA1429 helps stabilize Z-decay mRNAs in mouse oocytes [ 96 ]. Zc3h13 KD in mESCs significantly decreased global m 6 A level, and Zc3h13 KO resulted in morphological changes and reduced self-renewal ability of mESCs [ 41 ]. In zebrafish, mettl3 -null males and females have much lower rates of reproduction, and the sex ratio is impacted by the loss of mettl3 [ 23 ]. The establishment of a gonadal structure and function depends on the development of both germline and somatic cells [ 43 ]. Regarding gonadal development, in C . elegans , METT-10 (a METTL16 homolog) promotes vulva, somatic gonad, and embryo development, ensuring the differentiation of germ cells during meiosis [ 10 , 93 ]. The METTL3 and IME4 homologs in Drosophila melanogaster are mainly expressed in the testicles and ovaries, and Mettl3 is necessary for Notch signaling during oogenesis. Flies with Ime4 loss have a fatal phenotype [ 122 ]; however, recent research has indicated that m 6 A is necessary for female-specific alternative splicing, and that Drosophila Ime4 -null mutants are viable and fertile despite being unable to fly [ 22 , 24 ]. Successful blastocyst implantation is necessary for normal embryonic development after fertilization [ 123 ]. Decidua and placenta development must be closely coordinated during pregnancy to protect the fetus from maternal immune system attacks and to support fetal growth [ 124 ]. Many clinical conditions, such as preeclampsia and abortion, are associated with placental function. Changes in m 6 A modification also affect decidualization. The compromised postimplantation development of Mettl14 KO embryos may be due to defects in epiblast differentiation [ 121 ]. In Wtap mutant mice, the endoderm and ectoderm of the embryo did not develop normally [ 40 ]. Fto KO leads to closed chromatin in mESCs [ 104 ]. By up-regulating FLOT2, FTO causes granulosa cell dysfunction, which raises the possibility that FTO/FLOT2 may be involved in the pathogenesis of polycystic ovarian syndrome (PCOS) [ 125 ]. In a prior study, compared with normal individuals, the FTO level was lower and the m 6 A level was higher in women with spontaneous abortions [ 126 ]. Multiple abnormalities and severe developmental retardation can result from the loss of FTO function [ 127 ]. Embryos with FTO deletion show delayed development, and the maternal loss of FTO severely impedes decidua formation and embryo generation [ 104 ]. ALKBH5 KD in trophoblasts promotes trophoblast invasion. In contrast, the overexpression of ALKBH5 inhibits cell invasion [ 128 ]. In mice, preimplantation embryos, MII oocytes, and postnatal oocytes all have high levels of YTHDC1, while GV-stage oocytes have low levels [ 105 ]. Ythdc1 cKO mESCs exhibit markedly reduced proliferation rates [ 129 ]. Compared with those at E12.5, Ythdc2 expression was considerably higher in FGCs at E13.5 and E14.5. Cycloleucine-induced m 6 A inhibition or Ythdc2 KD in FGCs prevents meiotic entrance and prophase I progression [ 60 ]. Of two studies in zebrafish, one reported that removing ythdf2 prevented ZGA and accelerated the degradation of m 6 A-modified maternal mRNAs in zebrafish embryos [ 59 ]; the other study reported that the commencement of gastrulation, ZGA, and global maternal mRNA clearance were not impacted by the genetic deletion of the m 6 A readers Ythdf2 and Ythdf3 [ 13 ]. In summary, normal growth, sex determination, gonadal development, and zygotic gene activation are all impacted by m 6 A alteration ( Table 4 ). m 6 A modification is a dynamically changing process that plays an integral role in fertilization, embryonic development, embryo implantation, and postimplantation development. As shown by animal experiments and clinical trials, abnormalities in m 6 A modification and its modifiers lead to various development abnormalities. Understanding these processes may be helpful for drug research. Role of m 6 A and its regulators in embryonic development Note : ESC, embryonic stem cell; UTR, untranslated region; IME4, Inducer of MEiosis 4.

Epigenetics is the study of heritable changes that affect gene expression but do not result from DNA [ 1 ] or other nucleotide sequence alterations [ 2 ], such as DNA methylation, histone modification, chromatin rearrangement, and RNA modification, which are essential for controlling many physiological and pathological processes [ 3 ]. More than 160 structurally different RNA modifications have been identified in eukaryotes [ 4 , 5 ]. Methylation of bases and 2′-hydroxylation of RNA nucleotides are the most prevalent and straightforward RNA modifications [ 6 ]. N 6 -methyladenosine (m 6 A) is the most common form of messenger RNA (mRNA) modifications and was discovered in cancer cells as early as 1974 [ 7 , 8 ]. Each mRNA molecule exhibits, on average, between three and five sites of m 6 A modification [ 9 ]. With the advancement of epigenetics and the application of high-throughput sequencing technology, the biological functions and clinical applications of m 6 A have received increasing attention [ 10 ]. The biological effect of m 6 A is dynamically modulated by methyltransferases (writers), demethylases (erasers), and m 6 A-binding proteins (readers). Writers, encompassing a multicomponent methyltransferase complex, are responsible for adding m 6 A modifications [ 11 ]; erasers can remove m 6 A modifications [ 12 ]; and readers can recognize m 6 A modifications that mediate different downstream processes [ 13 ]. m 6 A modification typically occurs near exonic or intronic splice junctions and can therefore directly affect splicing [ 3 ]. It can modulate post-transcriptional gene expression by regulating pre-mRNA splicing and mRNA export, stability, and translation [ 14–19 ]. One of the most tightly controlled biological processes in a eukaryote’s life cycle is sexual reproduction, which is one of the most basic functions [ 20 ]. Germ cells encompass a special type of cell that can undergo meiosis. From late meiosis to early embryonic development, there is a period of gene transcriptional inactivity in the process of maternal-to-zygotic transition (MZT), and precise regulation of gene expression relies on post-transcriptional epigenetic modifications [ 21 ]. Hence, the m 6 A modification plays a crucial role in the initiation and progression of meiosis as well as in unique processes such as sperm–oocyte interactions and sex determination [ 22 ]. For example, defective gamete maturation has been associated with a reduction in the overall amount of m 6 A modifications [ 23 ], and only females have survived in the inducers of meiosis 4 ( Ime4 ) knockout (KO) fruit flies [ 22 , 24 ]. Impaired parental gamete formation or abnormal embryonic development of offspring will lead to infertility. In this review, we discuss the dynamic biological effects of the m 6 A modification and its regulators during gametic and embryonic development. In addition, we discuss drugs that can affect this modification and provide insights into strategies for the prevention and treatment of infertility at the epigenetic level.

Yujie Wang: Writing – original draft, Visualization. Chen Yang: Writing – review & editing. Hanxiao Sun: Writing – review & editing. Hui Jiang: Visualization. Pin Zhang: Supervision. Yue Huang: Supervision. Zhenran Liu: Supervision. Yaru Yu: Supervision. Zuying Xu: Supervision, Funding acquisition. Huifen Xiang: Funding acquisition, Writing – review & editing. Chengqi Yi: Conceptualization, Writing – review & editing. All authors have read and approved the final manuscript.

Changes in genetic material can lead to the development of many diseases. The secrets of epigenetics have gradually been revealed, explaining phenomena that heredity cannot explain. The transmission of epigenetic modifications is relatively stable during cell proliferation [ 180 ]. Drugs that can modify epigenetic processes, could be used to treat certain diseases. With advancements in epigenetic research, we may understand how the environment impacts fertility and devise mechanisms to prevent infertility. Studies have been conducted to derive male germ cells from pluripotent cells, and research is progressing to obtain oocytes from stem cells [ 181 , 182 ]. Since many procedures undertaken during in vitro fertilization (IVF) are conducted during the critical period of epigenetic recombination, including the removal of existing epigenetic modifications and replacement with new modifications in the somatic cell tissues of gametes and embryos [ 183 ], IVF may cause epigenetic alterations. Therefore, it is important to gain an in-depth understanding of the epigenetic changes occurring during IVF to reduce long-term complications in children. Many challenges still lie ahead. The mechanisms of some diseases are not clear, and a deeper understanding of these mechanisms is critical to the treatment of the disease. Research in other areas of study such as organoids, pluripotent stem cell differentiation, and synthetic biology [ 184 ], will further enhance our approach to understanding the mechanisms of infertility. Further in-depth research on the genetics and epigenetics of infertility can provide both short-term and long-term benefits. We will be able to improve the outcomes of pregnancy and the long-term health of the population through epigenetic treatment and promote the development of personalized drugs for fertility treatment. Creating in vitro models for spermatogenesis and oogenesis and investigating whether novel medications have an impact on gametogenesis are the best immediate focuses of clinical research [ 172 ].

Alterations in epigenetic modifications might be the focus of chemical therapeutic design and production to restore proper expression [ 6 , 130 , 131 ]. Inhibitors of m 6 A writers and erasers have been investigated. Due to the limited coverage of m 6 A modifications for reproductive health, we have summarized other aspects of m 6 A drugs. In 1997, METTL3 was identified as a key catalyst of the m 6 A modification [ 34 ]. METTL3 KD reduces methylation levels, accelerates apoptosis, reduces hyperplasia, and inhibits tumor growth in various cancers (acute myeloid leukemia; glioblastoma; uveal melanoma; osteosarcoma; oral, head and neck, and cutaneous squamous cell carcinoma; nasopharyngeal carcinoma; and breast, liver, bladder, gastric, prostate, lung, colorectal, pancreatic, thyroid, and ovarian cancer) [ 132–151 ]. The SAM simulator was the first METTL3 inhibitor discovered [ 152 ], but it has not been used clinically because of its poor cellular permeability and selectivity against other methyltransferases. Glioblastoma progression can be stopped by METTL3 overexpression in tumor cells or by pharmacologically inhibiting FTO demethylase [ 153 ]. Exon junction complexes are m 6 A inhibitors that protect proximal exon-binding RNA in the coding sequence from methylation and regulate mRNA stability through m 6 A inhibition [ 154 ]. Exon junction complexes are thought to be part of a new family of m 6 A regulators and suppressors that largely prevent the deposition of m 6 A [ 154 ]. Research continues on their ability to modulate gene expression outcomes. Nonspecific FTO inhibitors include the 2OG competitors N -oxalyl-glycine 1 and fumarate 2 [ 155 , 156 ], and FTO selective inhibitors include fluorescein derivatives FL2-DZ [ 157 ], a kind of compound [half-maximal inhibitory concentration (IC50) = 0.81 mM] [ 158 ], and entacapone [ 155 ]. Rhubaric acid-3 inhibits both the FTO and ALKB subfamilies [ 156 , 159 ]. A compound (IC50 = 8.7 mM) reduced FTO expression and showed potential usefulness in the treatment of epilepsy [ 160 ]. The FTO selective inhibitor MO-I-500 inhibited the survival and/or colony formation of triple-negative inflammatory breast cancer cell lines [ 161 , 162 ]. The FTO inhibitors R-2HG and FB23-2 can inhibit the growth of leukemia cells and slow the progression of leukemia; FB23-2 is more effective and can significantly inhibit acute myeloid leukemia progression in xenograft mice [ 163 , 164 ]. FTO regulates liver gluconeogenesis through the FOXO1 axis, and entacapone acts as an inhibitor of FTO, regulating fasting blood glucose through its direct effect on the liver FTO–FOXO1 signaling axis [ 165 ]. Studies on ALKBH5 inhibitors are limited. To date, both authorized medications and random substances with known activity have been investigated as m 6 A regulators [ 166 ]. The gut microbiome has a profound impact on a variety of elements of human health and disease, causing a variety of host reactions, including significant genetic changes. By lowering m 6 A levels in the intestinal mucosa of weaned piglets, resveratrol and curcumin could successfully enhance growth performance and maintain the integrity of the intestinal mucosa [ 167 ]. Thus, microorganisms can also affect the m 6 A modification level of mRNA in host tissues. In a clinical trial, the butyric acid levels in the digestive tracts of obese women with PCOS were lower than those in normal controls. Butyric acid reduces the expression of inflammatory cytokines by suppressing the expression of METTL3 , increasing ovarian function, and lowering the expression of local inflammatory factors in the ovaries [ 168 ]. The degree of global nucleic acid methylation, which includes DNA methylation and RNA m 6 A modification, was also lowered by exogenous vitamin C supplementation in immature swine Sertoli cells, increasing reproduction [ 169 ]. The m 6 A mutation in viral RNA helps viruses evade innate immunity [ 170 ]. Consequently, the application of m 6 A modification in the therapy of particular disorders is possible. These findings offer a fresh viewpoint for diagnosing and treating reproductive illnesses such as infertility.

RNA methylation is an important epigenetic modification. The dynamic changes in RNA m 6 A modification are caused by methyltransferases, demethylases, and m 6 A-binding proteins, and they affect various physiological and pathological processes, including spermatogenesis, oogenesis, embryogenesis, placental function, and human reproductive system diseases. Reproduction is one of the most strictly regulated key processes in the mammalian life cycle, and reproductive health is essential for human reproduction. Animal studies have shown that RNA m 6 A modification plays a crucial role in gametogenesis, embryonic development, and placental function. The theoretical foundation of germ cell development can be strengthened by understanding the functions of m 6 A regulators in gametogenesis and embryonic development, which could also help us uncover novel causes of infertility. As another important regulatory layer, RNA m 6 A modification in somatic cells of the reproductive system deserves extensive research. The regulation of RNA m 6 A modification imbalance can be used to restore function in the reproductive system and may help determine its core clinical value in infertility. m 6 A modification and its regulators can be targeted by drugs to treat certain diseases. Although this discovery has important theoretical and practical significance, it remains within the realm of basic research and has not yet undergone clinical translation.

With the rapid development of assisted reproductive technology, epigenetic research has helped promote the treatment of specific abnormalities; however, assisted reproductive technology induces epigenetic changes that could impair the development of fertilized mouse oocytes [ 171 ]. For example, during spermatogenesis, specific cells can be treated through epigenetic intervention. Sperm DNA is methylated differently in several maternal and paternal imprinted areas and shows a unique global methylation pattern [ 172 ]. During the gametogenesis and peri-implantation stages, reprogramming epigenome and imprinted loci, particularly imprinted loci, is important for maintaining the correct genetic pattern [ 172 ]. During germ cell development, histone deacetylation in oocyte maturation, energy metabolism, fertilization, mitochondrial function, genomic imprinting, and embryo genome activation are all involved in epigenetic inheritance [ 173 ]. When hormone stimulation fails to produce mature MII oocytes, in vitro maturation can be considered. Epigenetic regulation, such as histone acetylation and methylation, determines the quality of in vitro maturation [ 174 ]. As shown by genome sequencing technology, newborns conceived through assisted reproductive technology exhibit differences in methylation genes compared with those conceived naturally [ 175 , 176 ]. ALKBH5 mRNA expression in the chorionic tissue of patients with recurrent abortion is significantly increased, impairing the function of the trophoblastic layer [ 128 ]. Genetic susceptibility to PCOS is not only associated with alleles, but is also influenced by epigenetic changes [ 177 ]. Epigenetic aberrations have also been found in the ectopic endometrium of patients with endometriosis, and an understanding of the epigenome of the ectopic endometrium is key to understanding the genetic dysregulation and coordination that impair endometrial tolerance [ 178 , 179 ].

The authors have declared no competing interests.