Skip to main content

Epitranscriptome marks detection and localization of RNA modifying proteins in mammalian ovarian follicles



Most of the resources that support the early development of the embryo are stored in the oocyte. Clearing of maternal resources and activation of the embryonic genome to produce its own mRNA transcripts marks the maternal-to-embryo transition. Dependence on stored mRNA can last from a few hours to several days, depending on animal species. The mechanisms regulating stabilization and recruitment of stored maternal transcripts have not yet been described in full detail but are known to involve reversible polyadenylation and modulation of 3’UTR-mediated elements. RNA epigenetic modifications, new players in this field, have an important role in RNA regulation and stabilization.


The objectives of this study were first to determine if some of post-transcriptional methylation of stored mRNA is greater in oocytes than in somatic cells. We found that m6A, known to be the most prevalent and involved in various aspects of RNA metabolism and physiological functions, is particularly abundant in porcine oocyte mRNA compared to liver used as a somatic tissue reference. The second objective was to compare the epitranscriptome machinery, such as methyltransferases (“writers”), binding proteins (“readers”) and demethylases (“erasers”) catalyzing the different process, in follicles and oocytes of different mammalian species by immunofluorescence and confocal microscopy. The expression and localization patterns of these proteins differ between mice, pigs and cows ovaries and oocytes. m5C-associated proteins were generally less abundant. In contrast, m6A-associated proteins were expressed strongly during the early and late stages of folliculogenesis. Transzonal projections were found to contain more granules bearing the m5C mark in mice but both m5C and m6A methylation marks in association with mature oocytes of pigs and cows. Eraser proteins showed the greatest interspecies diversity in terms of distribution in the germinal tissues.


So far, few studies have looked at the oocyte and ovarian epitranscriptomic profile. Our findings indicate that a hitherto unrecognized species-specific layer of transcript regulation occurs at the RNA level and might be consequential during the oocyte transcriptional silencing period.


A central tenet of gene expression theory is that DNA-encoded information is first transcribed to RNA, an intermediary, transitory and short-lived molecule that is translated into protein as needed. However, oocytes display atypical RNA management, stocking molecules synthesized during oogenesis to support early development until embryonic genome activation [1]. Messenger RNA (mRNA) transcripts stored in oocytes exhibit an extended half-life that can be estimated in days rather than in minutes generally measured in somatic cells [2, 3].

Maternal RNA reserves ensure protein synthesis in the absence of transcriptional activity prior to genome activation [4, 5]. Transcriptional activity resumes differently depending on the species: 1-cell or 20 h post fertilization in mice [6], 4-cell or about 55 h post fertilization in pigs [7] and 8-cell or about 72 h post fertilization in cows [8,9,10,11].

The lifespan of maternal transcripts has not been measured directly but could in theory extend from final synthesis prior to DNA compaction in the oocyte to genome activation in the embryo. Compacted DNA is seen in oocytes still in the follicle. Based on the mean time for ovulation from this point plus fertilization and activation of embryo development, this lifespan can be estimated at 30 h in mice, 7 days in pigs and 10 days in cattle.

It is known that stabilization of maternal RNA involves removal of the poly(A) tail and packaging into ribonucleoprotein complexes [1]. In addition, specific sequences located in the 3’UTR have been shown to control stability [12]. The details of these mechanisms remain poorly understood and appear to differ greatly between species. Several RNA binding proteins and microRNA molecules have been shown to control the temporal destiny of transcripts but also the spatial distribution of mRNA in the oocyte cytoplasm [13, 14]. The influence of specific UTR sequences on maternal RNA stability is well documented, but spatial distribution has not been shown in mammalian oocytes [15].

Another type of post-transcriptional modification is RNA editing. Analogous to the epigenome, which is based primarily on 5-methylation of DNA cytidine groups (reviewed by Bird, 2002) [16], transcribed RNA is also modified by attachment of chemical groups [17]. Methylation is only one of more than 170 distinct chemical modifications that have been found on RNA molecules [17, 18]. The most abundant modification is N6-Methyladenosine (m6A), occurring mainly on mRNA and long non-coding RNA [19]. Enzymes that catalyze this reversible modification are called RNA modifying proteins or RMPs, of which three groups have been identified: writers, which catalyze the modification, erasers, which remove the marks, and readers, which recognize the modification and initiate a cellular response [20]. While the understanding of the function of the epitranscriptome has demonstrated its implication in various metabolic processes and diseases but its role in RNA stability and control of gene expression remains obscure.

We posit that some transcript labelling mechanism must exist and we hypothesized that different RNA methylation marks and process are involved in stabilizing stored maternal RNA. The aim of this study was therefore to compare the type and the abundance of RNA modifications in oocytes and somatic cells and to investigate if a relationship exists between the types of RMP present in oocytes of animals with different length of the transcriptional silence period. Modifications, namely m6A, were found abundantly in maternal RNA, as was the contingent of different RMPs, in some cases with species specific distributions.



All animals used in this study were handled according to the guidelines of the Canadian council on animal care and manipulation of animals used in research. Mouse experiments were carried out on 8-weeks old animals in accordance with protocols approved by the Comité de Protection des Animaux du Centre Hospitalier Universitaire de Québec. Bovine ovaries from 2–5 years old dairy cows and porcine ovaries and liver tissues from 5 months old gilts were collected from a local slaughterhouse.

All chemicals and enzymes, unless otherwise specified, were purchased from Sigma-Aldrich (Oakville, ON, Canada).

RNA isolation

Total RNA from oocytes, cumulus and granulosa cells were extracted with the Picopure RNA Isolation Kit (Thermo Fisher Scientific, Mississauga, ON, Canada) including on-column DNAse I digestion (Qiagen, Mississauga, ON, Canada) in accordance with the manufacturer’s instructions. Cumulus and granulosa cells were homogenized beforehand using QIAshredder spin-columns (Qiagen) and ovarian and liver tissues were homogenized using a Ruptor bead mill as recommended by the manufacturer (VWR International, Mississauga, ON, Canada). Total RNA was extracted from homogenates using TRIzol (Thermo Fisher Scientific) then isolated using a Picopure kit. Total RNA integrity and concentration were evaluated on a 2100-Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6000 Nano Kit (Agilent Technologies). Samples with a RIN greater than 7 were chosen.

RNA enzymatic digestion and LC–MS/MS analysis

RNA was converted to mono-ribonucleotides as described previously [21, 22]. Briefly, 1 µg of RNA was hydrolyzed for 3 h with 0.2 U of nuclease P1 in 60 µl of ammonium acetate buffer (pH 5.3) at 50 °C followed by 0.04 U of phosphodiesterase I (Thermo Fisher Scientific) for 2 h then 2 U of alkaline phosphatase for 2 h (both at 37 °C). Enzymes and buffers were removed using Nanosep 3 K columns (VWR International) centrifuged at 5000 × g for 30 min. Concentrated nucleotides were eluted in two 20 µl fractions of nuclease-free water for a total of 40 µl, dried using a speed-vac and kept at -80 °C until LC–MS/MS analysis as described previously [22]. Experiments were performed in triplicate. Experimental effects on nucleoside methylation were assessed by one-way ANOVA and the Tukey multiple comparisons test. Treatment means were considered significantly different when p < 0.05.

Ovary section immunohistofluorescence

Ovarian tissue (15 mm diameter, 5 mm thick) was fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS, Thermo Fisher Scientific) overnight at room temperature then dehydrated in ethanol at increasing concentrations and embedded in paraffin. Sections 7 µm thick were cut on a microtome, mounted in series on positively charged glass slides, dewaxed with xylene and rehydrated by washing with ethanol at concentrations decreasing to zero in distilled water. The slides were then processed in heat-induced epitope retrieval buffer (HIER, Agilent) in a pressure cooker at 95 °C for 20 min as per the manufacturer’s instructions (Biocare medical, Concord, CA, USA). Slides were cooled for about 5 min at room temperature then washed 3 times in PBS for 5 min each. Non‐specific binding was prevented by blocking with 5% donkey serum diluted in Tris-buffered saline with Tween 20 (TBST) for 1 h before applying the primary anti-rabbit or anti-chicken antibody diluted in TBST or the corresponding secondary antibody as a negative control (supplementary Fig. S1; antibodies are listed in supplementary Table S1). The slides were kept overnight at 4 °C in a humidified chamber then washed 3 times in TBST for 10 min each with gentle agitation and exposed for 1 h to the secondary antibody (diluted 1:1000 in TBST containing 20% bovine serum albumin) at room temperature with gentle agitation and shielding from light. DNA was stained using Hoechst 33,342 (Thermo Fisher Scientific). After three washes in PBS/TBST, slides were mounted in SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific).

Whole-mount oocyte immunofluorescence

Except for primary antibody applications, all steps below were performed at room temperature. Oocytes were fixed in 4% (v/v) paraformaldehyde in PBS-polyvinyl alcohol (PBS-PVA) for 15 min followed by three 5-min washes in PBS-PVA then permeabilized in Triton X-100 in TBST (1:200) for 15 min, blocked for 1 h with donkey serum (Sigma-Aldrich) diluted in TBST (1:20) and contacted with the primary antibody (in TBST with 20% BSA) overnight at 4 °C. Application of secondary antibody alone served as a negative control (supplementary Fig. S2). The oocytes were then washed 3 times in TBST (10 min each) and exposed for 1 h to secondary antibody (donkey anti-rabbit 488 or donkey anti-chicken 488, Thermo Fisher Scientific, diluted 1:1000 in TBST). DNA and actin (cytoskeleton) were stained respectively with Hoechst 33,342 (1:1000 in TBST) and Acti-Stain 555 (7:1000 in TBST) for 1 h. Oocytes were then washed 3 times in PBS-PVA (10 min each) and mounted in PBS-PVA.

Ovary and oocyte imaging

Z-stack images of oocytes and ovaries were captured using a Zeiss LSM700 Confocal microscope (Carl Zeiss Canada, Toronto, ON, Canada) fitted with a 40X water-immersion Zeiss objective and Zen Black acquisition software. The images were analyzed using Fiji ImageJ [23]. Secondary-only controls are shown in supplementary data. At least four ovary section slides and pools of 15 whole-mount oocytes from three different animals were examined.


Identification and measurement of RNA modifications by LC–MS/MS analysis

Methylated ribonucleosides, namely N1-methyladenosine, N6-methyladenosine, 5-methylcytosine and 7-methylguanosine (respectively m1A, m6A, m5C and m7G) in total RNA of oocytes, cumulus cells, granulosa cells, ovarian and liver tissue from pigs were detected quantitatively using LC–MS/MS. Liver was used as a somatic cell reference since mRNA is rarely stable in this tissue. As shown in Fig. 1 (A, B), RNA m1A was less abundant in oocytes than in liver, ovary and granulosa cells whereas m6A was more abundant in oocyte compared to liver. In the case of m5C and m7G, cumulus cells stand out with a low abundance of these molecules (Fig. 1C, D). In all tissues tested, the most abundant post-transcriptional modifications of RNA were m6A and m5C.

Fig. 1
figure 1

Relative expression of modifications of porcine somatic and germinal cell RNA, based on mass spectrometry (A) N1-methyladenosine, B N.6-methyladenosine, C 5-methylcytidine, D 7-methylguanosine. Means denoted by a different letter indicate significant differences between tissues (p < 0.05)

Detection of RNA modifying proteins

Using immunofluorescence and confocal microscopy, RMP writers, erasers and readers in follicles and oocytes from mice, porcine and bovine tissues were examined.

Detection of writer proteins during folliculogenesis

TRDMT1 (aka DNMT2) or tRNA aspartic acid methyltransferase 1 catalyzes methylation of cytosine. It is also slightly active as a DNA methyl transferase. This writer protein was barely detectable in early antral follicles of mice, pigs and cows and in mouse and cow oocytes (Fig. 2A and B). It was detected in the nucleus of porcine mature oocytes (Fig. 2B).

Fig. 2
figure 2

Representative confocal Z-stack images showing TRDMT1 and WTAP writer protein distribution (green) in murine, porcine and bovine germinal tissue obtained using fluorescent antibodies (green). A Sections of ovary showing secondary (S), early antral (Ea) and antral (A) follicle stages (bar = 20 μm). B Whole oocyte mounts (bar = 10 μm). Actin filaments of TZPs were stained with Acti-stain 555 phalloidin (red) and DNA with Hoechst 33,342 (blue)

In contrast, Wilms Tumor-Associated Protein 1 (WTAP), which produces m6A, was detected in mouse oocytes in pre-antral follicles, especially in the nucleus and around the zona pellucida. It appeared in the porcine oocyte nucleus (spots in Fig. 2A) and in follicular cells but not the nucleus of bovine oocytes (Fig. 2A, lower panel). During the later stages of follicular development, WTAP was found in the cytoplasm and nucleus of mature murine and bovine oocytes. In both large animals but not in mice, it was also localized throughout the network of transzonal projections (Fig. 2B, lower panel).

Detection of reader proteins

A family of proteins binds to methyl-CpG domains on DNA [24]. One of these, called Methyl-CpG Binding Domain Protein 2 (MBD2), has a high affinity for m5C-modified RNA [25, 26]. This protein was detected in the nucleus of early antral follicles in all species, but curiously only at the periphery of the oocyte nucleus in mice (Fig. 3A). It was more abundant both in the nucleus and the first layers of porcine cumulus cells and even more in the oocyte nucleus and cytoplasm, and in bovine cumulus cells. In fully mature oocytes, it was detected in the zona pellucida and at the end of transzonal projections in all three species, perhaps less in pigs (Fig. 3B).

Fig. 3
figure 3

Representative confocal Z-stack images of MBD2, YTHDF2 and FMRP reader protein distribution (green) in murine, porcine and bovine germinal tissue. A Sections of ovary showing primary (Pr), secondary (S) and early antral (Ea) follicle stages (bar = 20 μm). B Whole oocyte mounts (bar = 10 μm). Actin filaments of TZPs were stained with Acti-stain 555 phalloidin (red) and DNA with Hoechst 33,342 (blue)

Another reader protein, YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) member of the YTH domain family, recognize and bind to m6A (N6-methyladenosine) to regulate mRNA stability. YTHDF2 was distributed throughout the oocyte cytoplasm in all three species (Fig. 3A, middle panels) and was detectable in granulosa/cumulus cells and in the cytoplasm of mature murine or bovine but not porcine oocytes. Cumulus cells of all three species and the edge of murine and bovine zona pellucidae also contained YTHDF2, whereas transzonal protections contained very little (Fig. 3B, middle panels).

The RNA-binding protein FMRP (known as Fragile X Mental Retardation Protein or Fragile X Multifunctional RNA-binding Protein) is involved in shuttling transcripts and regulating their docking on ribosomes [27, 28]. Its binding is enhanced by m6A [29]. It was expressed strongly in early follicles of all three species, in mouse ovarian somatic cells and inside porcine and bovine oocyte follicular compartments as well as the oocyte cytoplasm (Fig. 3A). It was detected in antral follicles of all three species, in foci located under the zona pellucida and inside cumulus cells (Fig. 3B, lower panel). FMRP-containing granules were absent in murine and porcine transzonal projections but abundant near the cumulus side of the projections.

Detection of RNA methylation eraser proteins

Chemical marks imprinted on RNA by writer proteins can be erased by the action of demethylases such as AlkB homolog 5, RNA Demethylase (ALKBH5) or by FTO Alpha-Ketoglutarate Dependent Dioxygenase (FTO). In early follicles of all three species, ALKBH5 was found mostly in the oocyte nucleus (Fig. 4A). In antral follicles of mice, it appeared only in the gamete nucleus in a dotted pattern having no relationship to DNA (Fig. 4B). In the case of pigs and cows, it appeared in a diffuse state in the nuclear compartment but also in large foci in the subcortical region under the zona pellucida and was expressed strongly in cumulus cells. The distribution of FTO was broader, with a strong expression in the cytoplasm of early oocytes in all three species, finely dispersed in pig oocytes at a later follicular stage, and granular near the outer end of transzonal projections (the cumulus cell side of the zona pellucida) in pigs and cows. FTO granules inside transzonal projections were seen only in bovine oocytes. Table 1 provides a summary of the localization of the molecules detected.

Fig. 4
figure 4

Representative confocal Z-stack images of ALKBH5 and FTO eraser protein distribution (in green) in murine, porcine and bovine germinal tissue. A Sections of ovary showing primary (Pr), secondary (S) and early antral (Ea) and antral (An) follicle stages (bar: 20 μm). B Whole oocyte mounts (bar: 10 μm). Actin filaments of TZPs were stained with Acti-stain 555 phalloidin (red) and DNA with Hoechst 33,342 (blue)

Table 1 Localisation of RNA modifying proteins protein in early antral follicles and mature oocytes


Oocytes accumulate RNA reserves to sustain protein synthesis during the transcriptional silence that reigns from the moment the genome condenses in the immature oocyte until activation of the embryonic genome [5, 30]. The factors determining which stored mRNA transcripts are active during mammalian early development remain largely unknown but studies of Drosophila and Xenopus have shown clearly that mRNA is not stored randomly in the oocyte cytoplasm [31, 32]. Furthermore, it has been shown that maternal reserves need to be broken down for embryonic genome activation to occur [33, 34]. Their extended lifespan and active decay mechanisms in mice nevertheless indicate clearly that mRNA stability is crucial for constituting the reserves and seeing to their timely clearing.

The finding that m6A modification of RNA is more frequent in oocytes than in liver cells at least in pigs has interesting implications for maternal transcripts, since it has been demonstrated that an abundance of m6A near the stop codon in 3’UTRs affects polyadenylation site selection [35]. Another study in pigs has detected a greater abundance of m6A marks in the 5’ and 3’ UTR gene coding regions [36]. Moreover, it has found that the m6A is abundant in granulosa cells and this mark is greater in small (< 3 mm) compared to large follicles (> 5 mm) demonstrating a potential role of m6A during folliculogenesis through the modulation of specific transcript [36]. Since reproductive organs like ovaries exhibits a rate of aging that is much faster than other somatic, it has also been shown that m6A associated gene in granulosa cells from aging women, an increased number of m6A methylated genes compared to younger female group [37]. Differences in polyadenylation patterns have been associated with differences in transcript stability, export, translation and localization [38]. Transcriptional silencing of the X chromosome has been associated with the presence of at least 78 m6A marks throughout the long non-coding RNA of XIST [39]. Taken together these results provide novel insights of m6A in female fertility and its implication in various functions from transcript stability to folliculogenesis and ovarian functions through aging.

Just like on DNA, epigenomic marks on RNA are due to enzymatic activities that transfer, recognize or remove them. Some of these enzymes have been associated with infertility of one type or another. TRDMT1 (DNMT2) combined with NOL1/NOP2/SUN as partner enzymes has been shown to catalyze writing of m5C modifications [40]. Although we detected m5C by mass spectrometry, expression of TRDMT1 was very low in all species except for some in the nucleus of porcine oocytes. The presence of TRDMT1 in the nucleus was expected since it has CpG DNA methylation activity, which plays a key role in oogenesis and embryonic development [41, 42]. Cytosine methylation by TRDMT1 is important for the stabilization of tRNA [43]. In Drosophila, 5-hydroxymethylcytosine is found on polyadenylated RNA and promotes mRNA translation in different physiological processes including embryogenesis [44]. In the present study, WTAP was found in all three mammalian species. This m6A writer is known to play a role in embryogenesis and tissue differentiation in the zebra fish [45] through transcriptional and post-transcriptional regulation of various genes. It is localized in the nucleoplasm and in pre-mRNA-rich speckles, and co-localizes partially with splicing factors [45, 46]. In mice, wtap knock-out becomes lethal during early embryogenesis [47].

The reader proteins MBD2, YTHDF2 and FMRP were detected in the cytoplasm of early and mature oocytes in all three species. MBD2 proteins offer different localization in mice and bovine early embryo stages and MD2 knockout mice is not known to lead to gross abnormalities and animals remain fertile [24, 48]. However, YTHDF2 appears to be required for oocyte quality and zygotic development in mammals and regulates transcript dosage during oocyte maturation in mice [49]. YTHDF2 with YTHDC2 have been shown to be crucial for meiotic initiation and progression in mice female germ cells [50, 51]. FMR1 is located on the X chromosome and codes for an RNA-binding protein [52]. FMRP (the protein produced by FMR1) is considered as an m6A reader since it controls the export, the stability and the translation of methylated RNAs [29, 53,54,55]. Binding to m6A appears to be sequence-dependent, and possibly involve interaction with YTHDF [55]. FMR1 dysregulation is known to increase the risk of primary ovarian insufficiency leading to premature menopause [52]. How FMRP might cause primary ovarian insufficiency is still unclear, but its association is consistent with the role of RNA binding proteins that recognize methylation marks as essential elements of ovarian function.

The greatest differences between species were observed for eraser enzymes, both in terms of abundance and localisation. FTO was the most abundant in the oocyte cytoplasm and most abundant in mice. ALKBH5 and FTO are both essential for fertility, the former being involved in mRNA export and RNA metabolism as well as important for spermatogenesis [56] and the latter playing some role in preventing premature ovarian insufficiency disease [57]. It has also been shown that both are localized in the nucleus of eukaryotic cells [19, 58], although we found that FTO was most abundant in the oocyte cytoplasm. FTO can also be an eraser for N6,2’-O-dimethyladenosine (m6Am), a post-transcriptional modification that occurs on the first nucleotide on mRNA following the m7G cap [59]. The reversible m6Am mark stabilizes mRNA [60]. Our observations of nucleoside modifications and the abundance of the associated RMPs are consistent with this important role of m6A during oogenesis. The differences between species in terms of FTO are consistent with the timing required for the breakdown of maternal reserves.

We have reported previously that in cattle, transzonal projections connecting cumulus cells to the oocyte act like synapses and can harbor ribonucleoprotein granules or transfer these and other complex materials to the oocyte [61, 62]. RNA thus transferred would add to the maternal reserves. In this study, we showed that m6A writer WTAP is detectable in transzonal projections of both large mammals but not in those of mice. The three readers were found inside projections in all three species, whereas FTO was only in the case of cattle. This is consistent with observed cross-species differences in the duration of transzonal attachment to the oolemma, for example, persisting during maturation in mice, rats, rabbits and humans but disconnecting and retracting immediately after germinal vesicle breakdown in cattle [63].

The mechanisms underlying RNA shuttling through transzonal projections are still largely unknown, but we and others have described the role played by FMRP in RNA granules transport in mouse neurons [28]. N6-methyladenosine pulldown has allowed identification of FMRP, FXR1P and FXR2P as new readers [64]. Moreover, in mouse embryonic stem cells, FMRP also shares targets with YTHDF1 and promotes nuclear export of transcripts containing m6A residues [29, 64]. These reports all corroborate the involvement of RNA modifying enzymes in transcript stability, transport and translational control. 


Three animal species were compared in an attempt to reveal the role of methylation in the persistence of maternal mRNA during mammalian early development. The longest transcriptional silence period and the earliest disconnection of transzonal projections following meiosis resumption are observed in bovine oocytes. Murine oocytes have the shortest transcriptional silence and are the least dependent on transcript stability and storage. Known modifiers of RNA nucleoside bases were very abundant in oocytes of all three species from the early to antral follicle stages. Species-associated differences in the distribution of these enzymes were observed, notably for the m6A eraser FTO. Increased presence of RNA modifying enzymes in transzonal projections was associated with greater dependence on RNA stability. This study provides support for an important role played by the epitranscriptome in the management of maternal RNA stored in mammalian oocytes.

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.



AlkB homolog 5, RNA Demethylase


Fragile X Messenger Ribonucleoprotein 1


Fragile X Mental Retardation Protein or Fragile X Multifunctional RNA-binding Protein)


FTO Alpha-Ketoglutarate Dependent Dioxygenase


Liquid Chromatography with tandem mass spectrometry


Messenger RNA










Methyl-CpG Binding Domain Protein 2


RNA modifying protein


TRNA aspartic acid methyltransferase 1


Untranslated region


Wilms Tumor-Associated Protein 1


YTH N6-Methyladenosine RNA Binding Protein F2


  1. Bachvarova RF. A maternal tail of poly(A): the long and the short of it. Cell. 1992;69:895–7.

    Article  CAS  PubMed  Google Scholar 

  2. Jahn CL, Baran MM, Bachvarova R. Stability of RNA synthesized by the mouse oocyte during its major growth phase. J Exp Zool. 1976;197:161–71.

    Article  CAS  PubMed  Google Scholar 

  3. Raghavan A. Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res. 2002;30:5529–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Memili E, First NL. Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote. 2000;8:87–96.

    Article  CAS  PubMed  Google Scholar 

  5. Lodde V, Modina S, Maddox-Hyttel P, Franciosi F, Lauria A, Luciano AM. Oocyte morphology and transcriptional silencing in relation to chromatin remodeling during the final phases of bovine oocyte growth. Mol Reprod Dev. 2008;75:915–24.

    Article  CAS  PubMed  Google Scholar 

  6. Schultz RM. Regulation of zygotic gene activation in the mouse. BioEssays News Rev Mol Cell Dev Biol. 1993;15:531–8.

    Article  CAS  Google Scholar 

  7. Mateusen B, Van Soom A, Maes DGD, Donnay I, Duchateau L, Lequarre A-S. Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study. Reprod Camb Engl. 2005;129:443–52.

    CAS  Google Scholar 

  8. Barnes FL, First NL. Embryonic transcription in in vitro cultured bovine embryos. Mol Reprod Dev. 1991;29:117–23.

    Article  CAS  PubMed  Google Scholar 

  9. Frei RE, Schultz GA, Church RB. Qualitative and quantitative changes in protein synthesis occur at the 8–16-cell stage of embryogenesis in the cow. J Reprod Fertil. 1989;86:637–41.

    Article  CAS  PubMed  Google Scholar 

  10. Kopecny V. High-resolution autoradiographic studies of comparative nucleologenesis and genome reactivation during early embryogenesis in pig, man and cattle. Reprod Nutr Dév. 1989;29:589–600.

    Article  CAS  PubMed  Google Scholar 

  11. Anderson JE, Matteri RL, Abeydeera LR, Day BN, Prather RS. Degradation of maternal cdc25c during the maternal to zygotic transition is dependent upon embryonic transcription. Mol Reprod Dev. 2001;60:181–8.

    Article  CAS  PubMed  Google Scholar 

  12. Esencan E, Kallen A, Zhang M, Seli E. Translational activation of maternally derived mRNAs in oocytes and early embryos and the role of embryonic poly(A) binding protein (EPAB). Biol Reprod. 2019;100:1147–57.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Irion U, Adams J, Chang C-W, St JD. Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway. Dev Biol. 2006;297:522–33.

    Article  CAS  PubMed  Google Scholar 

  14. Tripurani SK, Wee G, Lee K-B, Smith GW, Wang L. MicroRNA-212 post-transcriptionally regulates oocyte-specific basic-helix-loop-helix transcription factor, factor in the germline alpha (FIGLA), during bovine early embryogenesis. PLoS ONE. 2013;8: e76114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Calder MD, Madan P, Watson AJ. Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins. Zygote. 2008;16:161–8.

    Article  CAS  PubMed  Google Scholar 

  16. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 2002;16:6–21.

    Article  CAS  PubMed  Google Scholar 

  17. Fu Y, Dominissini D, Rechavi G, He C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat Rev Genet. 2014;15:293–306.

    Article  CAS  PubMed  Google Scholar 

  18. Boccaletto P, Stefaniak F, Ray A, Cappannini A, Mukherjee S, Purta E, MODOMICS: a database of RNA modification pathways., et al. update. Nucleic Acids Res. 2021;2022(50):D231–5.

    Google Scholar 

  19. Du H, Zhao Y, He J, Zhang Y, Xi H, Liu M, et al. YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4–NOT deadenylase complex. Nat Commun. 2016. [cited 2019 Aug 27];7. Available from:

  20. Esteve-Puig R, Bueno-Costa A, Esteller M. Writers, readers and erasers of RNA modifications in cancer. Cancer Lett. 2020;474:127–37.

    Article  CAS  PubMed  Google Scholar 

  21. Yan M, Wang Y, Hu Y, Feng Y, Dai C, Wu J, et al. A high-throughput quantitative approach reveals more small RNA modifications in mouse liver and their correlation with diabetes. Anal Chem. 2013;85:12173–81.

    Article  CAS  PubMed  Google Scholar 

  22. Fu L, Amato NJ, Wang P, McGowan SJ, Niedernhofer LJ, Wang Y. Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method. Anal Chem. 2015;87:7653–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.

    Article  CAS  PubMed  Google Scholar 

  24. Ruddock-D’Cruz NT, Xue J, Wilson KJ, Heffernan C, Prashadkumar S, Cooney MA, et al. Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development. Mol Reprod Dev. 2008;75:48–59.

  25. Courtney DG, Tsai K, Bogerd HP, Kennedy EM, Law BA, Emery A, et al. Epitranscriptomic Regulation of HIV-1 Gene Expression by m 5C and the Novel m 5C Reader MBD2. SSRN Electron J. 2019. [cited 2022 Aug 30]; Available from:

  26. Kong W, Biswas A, Zhou D, Fiches G, Fujinaga K, Santoso N, et al. Nucleolar protein NOP2/NSUN1 suppresses HIV-1 transcription and promotes viral latency by competing with Tat for TAR binding and methylation. PLoS Pathog. 2020;16: e1008430.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Khandjian EW, Corbin F, Woerly S, Rousseau F. The fragile X mental retardation protein is associated with ribosomes. Nat Genet. 1996;12:91–3.

    Article  CAS  PubMed  Google Scholar 

  28. El Fatimy R, Davidovic L, Tremblay S, Jaglin X, Dury A, Robert C, et al. Tracking the Fragile X Mental Retardation Protein in a Highly Ordered Neuronal RiboNucleoParticles Population: A Link between Stalled Polyribosomes and RNA Granules. Darnell JC, editor. PLOS Genet. 2016;12:e1006192.

  29. Hsu PJ, Shi H, Zhu AC, Lu Z, Miller N, Edens BM, et al. The RNA-binding protein FMRP facilitates the nuclear export of N 6-methyladenosine-containing mRNAs. J Biol Chem. 2019;294:19889–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kloc M, Bilinski S, Chan AP, Allen LH, Zearfoss NR, Etkin LD. RNA localization and germ cell determination in Xenopus. Int Rev Cytol. 2001;203:63–91.

    Article  CAS  PubMed  Google Scholar 

  31. Becalska AN, Gavis ER. Lighting up mRNA localization in Drosophila oogenesis. Dev Camb Engl. 2009;136:2493–503.

    CAS  Google Scholar 

  32. Alizadeh Z, Kageyama S-I, Aoki F. Degradation of maternal mRNA in mouse embryos: Selective degradation of specific mRNAs after fertilization. Mol Reprod Dev. 2005;72:281–90.

    Article  CAS  PubMed  Google Scholar 

  33. Schultz RM, Stein P, Svoboda P. The oocyte-to-embryo transition in mouse: past, present, and future. Biol Reprod. 2018;99:160–74.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ke S, Alemu EA, Mertens C, Gantman EC, Fak JJ, Mele A, et al. A majority of m 6 A residues are in the last exons, allowing the potential for 3′ UTR regulation. Genes Dev. 2015;29:2037–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cao Z, Zhang D, Wang Y, Tong X, Avalos LFC, Khan IM, et al. Identification and functional annotation of m6A methylation modification in granulosa cells during antral follicle development in pigs. Anim Reprod Sci. 2020;219: 106510.

    Article  CAS  PubMed  Google Scholar 

  36. Liu C, Li L, Yang B, Zhao Y, Dong X, Zhu L, et al. Transcriptome-wide N6-methyladenine methylation in granulosa cells of women with decreased ovarian reserve. BMC Genomics. 2022;23:240.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Tian B, Manley JL. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol. 2017;18:18–30.

    Article  CAS  PubMed  Google Scholar 

  38. Patil DP, Chen C-K, Pickering BF, Chow A, Jackson C, Guttman M, et al. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature. 2016;537:369–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bohnsack K, Höbartner C, Bohnsack M. Eukaryotic 5-methylcytosine (m5C) RNA Methyltransferases: Mechanisms, Cellular Functions, and Links to Disease. Genes. 2019;10:102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992;69:915–26.

    Article  CAS  PubMed  Google Scholar 

  41. Uysal F, Akkoyunlu G, Ozturk S. Dynamic expression of DNA methyltransferases (DNMTs) in oocytes and early embryos. Biochimie. 2015;116:103–13.

    Article  CAS  PubMed  Google Scholar 

  42. Tuorto F, Liebers R, Musch T, Schaefer M, Hofmann S, Kellner S, et al. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat Struct Mol Biol. 2012;19:900–5.

    Article  CAS  PubMed  Google Scholar 

  43. Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, et al. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science. 2016;351:282–5.

    Article  CAS  PubMed  Google Scholar 

  44. Ping X-L, Sun B-F, Wang L, Xiao W, Yang X, Wang W-J, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24:177–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10:93–5.

    Article  CAS  PubMed  Google Scholar 

  46. Horiuchi K, Umetani M, Minami T, Okayama H, Takada S, Yamamoto M, et al. Wilms’ tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA. Proc Natl Acad Sci. 2006;103:17278–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hendrich B, Guy J, Ramsahoye B, Wilson VA, Bird A. Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development. Genes Dev. 2001;15:710–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ivanova I, Much C, Di Giacomo M, Azzi C, Morgan M, Moreira PN, et al. The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence. Mol Cell. 2017;67:1059-1067.e4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhao H, Zhou D, Chen Y, Liu D, Chu S, Zhang S. Beneficial effects of Heqi san on rat model of polycystic ovary syndrome through the PI3K/AKT pathway. Daru J Fac Pharm Tehran Univ Med Sci. 2017;25:21.

    Article  Google Scholar 

  50. Zeng M, Dai X, Liang Z, Sun R, Huang S, Luo L, et al. Critical roles of mRNA m6A modification and YTHDC2 expression for meiotic initiation and progression in female germ cells. Gene. 2020;753: 144810.

    Article  CAS  PubMed  Google Scholar 

  51. Garber KB, Visootsak J, Warren ST. Fragile X syndrome. Eur J Hum Genet. 2008;16:666–72.

    Article  CAS  PubMed  Google Scholar 

  52. Zhang F, Kang Y, Wang M, Li Y, Xu T, Yang W, et al. Fragile X mental retardation protein modulates the stability of its m6A-marked messenger RNA targets. Hum Mol Genet. 2018;27:3936–50.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Edens BM, Vissers C, Su J, Arumugam S, Xu Z, Shi H, et al. FMRP Modulates Neural Differentiation through m6A-Dependent mRNA Nuclear Export. Cell Rep. 2019;28:845-854.e5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Worpenberg L, Paolantoni C, Longhi S, Mulorz MM, Lence T, Wessels H-H, et al. Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila. EMBO J. 2021;40: e104975.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang C-M, Li CJ, et al. ALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility. Mol Cell. 2013;49:18–29.

    Article  CAS  PubMed  Google Scholar 

  56. Ding C, Zou Q, Ding J, Ling M, Wang W, Li H, et al. Increased N6-methyladenosine causes infertility is associated with FTO expression. J Cell Physiol. 2018;233:7055–66.

    Article  CAS  PubMed  Google Scholar 

  57. Yang Y, Hsu PJ, Chen Y-S, Yang Y-G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res. 2018;28:616–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Keith JM, Ensinger MJ, Moss B. HeLa cell RNA (2’-O-methyladenosine-N6-)-methyltransferase specific for the capped 5’-end of messenger RNA. J Biol Chem. 1978;253:5033–9.

    Article  CAS  PubMed  Google Scholar 

  59. Mauer J, Luo X, Blanjoie A, Jiao X, Grozhik AV, Patil DP, et al. Reversible methylation of m6Am in the 5’ cap controls mRNA stability. Nature. 2017;541:371–5.

    Article  CAS  PubMed  Google Scholar 

  60. Macaulay AD, Gilbert I, Caballero J, Barreto R, Fournier E, Tossou P, et al. The Gametic Synapse: RNA Transfer to the Bovine Oocyte1. Biol Reprod. 2014;91(4):90.

    Article  PubMed  Google Scholar 

  61. Macaulay AD, Gilbert I, Scantland S, Fournier E, Ashkar F, Bastien A, et al. Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation. Biol Reprod. 2016;94:16.

    Article  PubMed  Google Scholar 

  62. Thibault, Charles, Levasseur, Marie-Claire. La reproduction chez les mammifères et l’homme. Nouvelle édition entièrement refondue. Paris: Ellipses; 2001.

  63. Edupuganti RR, Geiger S, Lindeboom RGH, Shi H, Hsu PJ, Lu Z, et al. N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis. Nat Struct Mol Biol. 2017;24:870–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Xiao S, Duncan FE, Bai L, Nguyen CT, Shea LD, Woodruff TK. Size-specific follicle selection improves mouse oocyte reproductive outcomes. Reprod Camb Engl. 2015;150:183–92.

    CAS  Google Scholar 

  65. Tan J-H, Wang H-L, Sun X-S, Liu Y, Sui H-S, Zhang J. Chromatin configurations in the germinal vesicle of mammalian oocytes. Mol Hum Reprod. 2009;15:1–9.

    Article  CAS  PubMed  Google Scholar 

  66. Bui H-T, Wakayama S, Kishigami S, Kim J-H, Van Thuan N, Wakayama T. The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming. Dev Camb Engl. 2008;135:3935–45.

    CAS  Google Scholar 

  67. Luciano AM, Franciosi F, Dieci C, Lodde V. Changes in large-scale chromatin structure and function during oogenesis: a journey in company with follicular cells. Anim Reprod Sci. 2014;149:3–10.

    Article  CAS  PubMed  Google Scholar 

  68. El Fatimy R, Tremblay S, Dury AY, Solomon S, De Koninck P, et al. Fragile X mental retardation protein interacts with the RNA-binding protein Caprin1 in neuronal RiboNucleoProtein complexes [corrected]. PLoS One. 2012;7(6):e39338.

    Article  PubMed  PubMed Central  Google Scholar 

Download references


The authors want to thank Isabelle Laflamme for her technical expertise and support.


The authors acknowledge the following funding agencies for their support: Natural Sciences and Engineering Research Council of Canada (Grant RGPIN-2017–04775) and Fonds Québécois de la Recherche sur la Nature et les Technologies (Grant 182922).

Author information

Authors and Affiliations



Claude Robert designed and conceived the project. Karine Dubuc performed the experiments with the help of Malthilde Marchais, Isabelle Gilbert, Alexandre Bastien and Karen Nenonene. Claude Robert and Karine Dubuc analysed and interpreted the data with the help of Edward Khandjian, Robert Viger and Géraldine Delbes. Karine Dubuc, Isabelle Gilbert and Claude Robert wrote the manuscript. All authors read, revised and approved the manuscript.

Corresponding author

Correspondence to Claude Robert.

Ethics declarations

Ethics approval and consent to participate

Experimental protocols in this study were performed in accordance with the Canadian council on animal care and manipulation of animals used in research. Mice experiments were approved by the Comité de Protection des Animaux du CHU de Québec.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Table S1. Description of the antibodies used for immunofluorescence in this study.

Additional file 2:

Figure S1. Ovarian section in paraffin, negative control without primary antibody, showing no specific staining by the secondary antibody. DNA is stained with Hoescht 33342 (blue). Bar = 20 µm.

Additional file 3:

Figure S2. Oocyte whole mount, negative control without primary antibody, showing no specific staining by the secondary antibody. DNA is stained with Hoescht 33342 (blue) and actin with Acti-stain 555 phalloidin (red). Bar = 20 µm (same magnification was used for swine and bovine).

Additional file 4:

Stage and diameter of the follicles used in this study and the corresponding chromatin stage and transcription activity reported in the literature[65,66,67,68].

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dubuc, K., Marchais, M., Gilbert, I. et al. Epitranscriptome marks detection and localization of RNA modifying proteins in mammalian ovarian follicles. J Ovarian Res 16, 90 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • RNA methylation
  • Oocytes
  • Transzonal projections
  • Somatic cells
  • RNA modifying enzyme
  • Epitranscriptome