Fertilization produces totipotent cells differentiating into any tissue via zygote formation mammals. After fertilization, maternal RNA is degraded and new RNA is transcribed, usually known as maternal-to-zygotic transition (MZT) (Tadros and Lipshitz, 2009). In different species, zygotic genome activation (ZGA) timing for early embryo development differs at the embryonic division stage (2-cells in mice, 4-cells in pigs, and 8-cells in humans and cattle) (Telford et al., 1990; Latham et al., 1991; Aoki et al., 1997; Hyttel et al., 2000). ZGA triggers the global gene expression initiation to prepare the differentiation into any cells or tissues and development into a complete organism. During ZGA, new RNAs are synthesized through pre- and post-transcriptional control, acquired for cell fate determination and further development. Pre-transcriptional controls such as chromatin remodeling, histone modification, and DNA methylation changes regulates progressively acquired specific gene expression patterns essential for embryonic development (Kashyap et al., 2009; Hanna et al., 2010; Apostolou and Hochedlinger, 2013; Burton and Torres-Padilla, 2014). Currently, post-transcriptional controls such as transcribed RNA export, capping, stability, poly-A tailing, and splicing is necessary for the key regulation of gene expression (Ulitsky et al., 2012; Zhao et al., 2017). Recent results suggest that modifications of mRNAs, including m⁶A, polyadenylation (Poly-A), and uridine tail (U-Tail) formation, play significant roles in mRNA regulation (Chang et al., 2014), but its functional roles and relationship on early embryo development have been elusive.

N-6 methyl adenosine (m⁶A or RNA methylation) is currently identified as a novel post-transcriptional regulator in eukaryotic cells (Desrosiers et al., 1975; Perry et al., 1975; Wei et al., 1975). It has been detected in specific positions in introns, exons, and untranslated regions (UTRs) of more than 7,000 mRNA and 300 non-doing RNA (ncRNA) in humans and is installed by the writers such as METTL3 and METTl14 and erasers such as FTO or ALKBH5 (Bokar et al., 1997; Fawcett and Barroso, 2010; Zheng et al., 2013; Liu et al., 2014). METTL3 is crucial formouse embryonic stem cells and embryo fate determination, while ALKBH5 is crucial for male mouse fertility by altering gene expression patterns involving spermatogenesis (Batista et al., 2014; Tang et al., 2018). Thus, specific gene expression regulation by these m⁶A effector proteins has major biological functions particularly in development. m⁶A binding protein YTHDF2 is required for oocyte maturation and early zygotic development by regulating maternal RNA (Zhao et al., 2017). Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), another m⁶A reader protein, belongs to an RNA-binding protein family that contributes to various transcription control mechanisms, such as alternative splicing, poly-A tailing, mRNA stability, export, and translational control. hnRNPA2/B1 has the functional roles of pre-mRNA processing with sequence specificity in cells (Beyer et al., 1977; Dreyfuss et al., 1993). Moreover, hnRNPA2/B1 depletion affects chicken embryo development by interrupting smooth muscle differentiation (Wang et al., 2012). Human embryonic stem cells are also regulated by hnRNPA2/B1 during G1/S transition and involves self-renewal programs (Choi et al., 2013). However, the roles of hnRNPA2/B1 during porcine early embryo development are not well known.

In light of the importance of hnRNPA2/B1 in these mRNA processing and development we investigated the effect of hnRNPA2/B1 on embryogenesis using RNA interference (RNAi) approach. To the best of our knowledge, this is the first study to investigate the spatial and temporal expression pattern and the biological function of hnRNPA2/B1 in porcine pre-implantation embryo development.

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.

Porcine embryo collection and in vitro maturation

Porcine pre-pubertal ovaries were obtained from a nearby local slaughterhouse, and transported to the laboratory in pre-warmed saline supplemented with 75 mg/mL penicillin G and 50 mg/mL streptomycin sulfate. After washing the ovaries with Tyrode’s lactate HEPES polyvinyl alcohol (TL-HEPES) medium 15, the follicles were aspirated using an 18-gauge needle with 10 mL syringe. Cumulus oocyte complexes (COCs) deposited at the bottom were resuspended with HEPES-PVA and the high density COCs were collected on the heated stage adjusted to 38.5℃. For in vitro oocyte maturation, the collected oocytes were transferred into 4-well dish with porcine in vitro maturation medium comprising TCM-199 (11150-059, Thermo Fisher Scientific; Waltham, MA, USA) containing 0.57 mM cysteine, 0.91 mM NA-pyruvate, 75 μg/mL Kanamycin, 1 μg/mL Insulin, 20 ng/mL EGF, 10 ng/mL Leptin, 10 IU/mL FSA, 10 IU/mL LH and 10% of filtered porcine follicle fluid (PFF). And then, COCs were incubated at 38.5℃ in a humidified 5% CO₂ atmosphere, and maturated MII oocytes were used for parthenogenetic activation.

Parthenogenetic activation and embryo development in vitro

Polar body extruded MII oocytes were denuded by 1 mg/mL hyaluronidase with rigorous pipetting. After gradually adapting to high concentration mannitol solution (ddH₂O with 297 mM mannitol, 0.1 mM CaCl₂⸱2H₂O, 0.05 mM MgCl₂⸱6H₂O, 280 mOsm/L, and pH 7.2), the oocytes are finally activated through two electrical stimulations (120 V for 60 μs) using Electrocell Manipulator (BTX; San Diego, CA, USA). The activated embryos were exposed to 7.5 mg/mL Cytocalasin B for 3 H in porcine zygote medium-5 (PZM-5) supplemented with 0.4% bovine serum albumin (BSA) and incubated at 38.5℃ in a humidified 5% CO₂ atmosphere.

Double stranded RNA (dsRNA) injection and embryo collection

hnRNPA2/B1 was knocked down in porcine parthenotes via microinjecting target gene double stranded RNA (dsRNA) after parthenogenetic activation. hnRNPA2/B1 dsRNA was designed from the T7 promoter sequence containing sequence deposited in NCBI database (XM_021078978.1) and microinjected into the zygote cytoplasm using an Eppendorf FemtoJet (Eppendorf; Hamburg, Germany) and a Nikon TE2000-U inverted microscopy (Nikon Corporation; Kokyo, Japan). The control embryos were infected with eGFP dsRNA and then cultured in PZM-5 for 144 h.

Immunocytochemistry (ICC)

Porcine embryos were washed in Dulbecco’s phosphate-buffered saline (DPBS) containing 1 mg/mL polyvinyl alcohol (PBS-PVA), fixed for 30 min at room temperature in 3.7% (w/v) paraformaldehyde dissolved in DPBS, permeabilized with 0.2% (v/v) Triton X-100 in DPBS for 1 h, and blocked with 3% BSA in DPBS (PBS-BSA). The embryos were incubated with first antibody (hnRNPA2/B1: mouse polyclonal, ab6102, Abcam) overnight at 4℃ and then with an FITC-conjugated secondary antibody (Invitrogen). Finally, the nuclei were stained with Hoechst 33342 (Sigma-Aldrich) and mounted on a glass slide with Vectashield. Between every step, the embryos were rinsed in PBS-PVA thrice for 10 min at room temperature. The stained embryos were examined under a confocal microscope (Zeiss LSM 710 Meta). The images were processed using ImageJ software.

Real time reverse-transcription polymerase chain reaction (real time RT-PCR)

The embryos were washed in PBA-PBS thrice, frozen with lysis buffer of Dynabeads mRNA Direct Kit (Dynal Asa; Oslo, Norway) in liquid nitrogen, and stored at -80℃ before extracted the mRNA. cDNA was synthesized from isolated mRNA using 1^st strand cDNA synthesis kit (Legene Bioscience; San Diego, CA, USA). The mRNA expression of several genes was then detected by real time RT-PCR (qRT-PCR) with specific primer pairs (Table 1). Quantitative RT-PCR was performed on a Bio-rad real time PCR instrument using the DyNAmo SYBR Green qPCR Kit containing modified hot-start Tbr DNA polymerase, SYBR Green, an optimized PCR buffer, 5 mM MgCl₂, and a dNTP mix with dUTP (Finnzymes Oy, Espoo, Finland) according to the instructions of the machine and kit manufacturers. PCR was performed as follows: denaturation at 95℃ for 10 min, 40 cycles of amplification and quantification at 94℃ for 10 s, 60℃ for 30 s, and 72℃ for 30 s with a single fluorescence measurement, melting at 65-95℃ with 0.2℃/s heating rate, and continuous fluorescence measurement and cooling to 12℃. The PCR products were analyzed by generating melting curves to ensure gene-specific amplifications. The relative quantification of gene expression was determined by the 2^-ΔΔCt method. GAPDH was used as an internal control in all experiments and primer information are listed in Table 1.

Quantification of m⁶A RNA methylation

Total RNA was isolated from 30 of blastocysts using TRIzol and m⁶a RNA methylation was quantified using used m⁶A RNA quantification kit (Epigentek, NY, UA). 200 ng of control and hnRNPA2/B1 KD blastocysts RNA were added with binding solution in each strip well and incubate at 37 for 90 min. To m⁶A RNA capture, 1^st capture antibody (1:1,000) and 2^nd capture antibody (1:2,000) (1:2,000) antibody were treated following incubation at RT for 60 min. And then, 1:5,000 enhancer buffer at RT 30 min. Washing step with 1X wash buffer carried out between each step. Detection solution was added in each wells and incubated in RT for 10 min without light. Finally, m⁶A RNA was read on microplate reader Sunrise (Tecan, Männedorf Switzerland) ant 450 nm.

Statistical analysis

Data were analyzed by independent sample t-test using GraphPad Prism 8 software (GraphPad; San Diego, CA, USA) and each experiment was repeated at least thrice. Data are presented as mean ± standard error mean (S.E.M). p < 0.05 were considered statistically significant unless otherwise stated.

Characterization of hnRNPA2/B1 during porcine parthenotes

To confirm HnRNPA2/B1 mRNA expression pattern and localization during pre-implantation development, we carried out the qRT-PCR and immunocytochemistry (ICC). HnRNPA2/B1 localization was evenly detected in the nuclei and cytoplasm, except in 1-cell nuclei (Fig. 1A). Then, we checked the mRNA levels during embryogenesis. At the 2-cell stage, hnRNPA2/B1 mRNA levels increased, peaked from 2-cell to 4-cell stage, and subsequently decreased until blastocyst stage (p < 0.01; Fig. 1B). These results revealed that hnRNPA2/B1 mRNA transcription initiated before ZGA, while being localized at the nuclei during porcine early embryo development.

Figure 1.Expression patterns of hnRNPA2/B1 in porcine embryogenesis. HnRNPA2/B1 mRNA expression and localization of hnRNPA2/B1 in porcine pre-implantation embryos. (A) Immunocytochemistry (ICC) showed that hnRNPA2/B1 is found in the nuclei and cytoplasm during porcine pre-implantation embryo development. (B) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of hnRNPA2/B1 in 1-cell (1C), 2-cell (2C), 4-cell (4C), morula and the blastocyst. Error bars indicate mean ± standard error of the mean (S.E.M). Scale bars = 50 μM.

Effect of hnRNPA2/B1 knock-down during porcine early embryo development

Next, we investigated the function of hnRNPA2/B1 in porcine early embryo by dsRNA microinjection into zygote cytoplasm. Porcine hnRNPA2/B1 transcript and protein levels were significantly reduced after hnRNPA2/B1 knock-down (Fig. 2A-C). The blastocyst development rates in hnRNPA2/B1 knock-down embryos were significantly decreased than that in control embryos (Fig. 2D and 2E). These results show that hnRNPA2/B1 knock-down inhibited blastocyst formation.

Figure 2.Effects of RNAi-mediated hnRNPA2/B1 knock-down on porcine pre-implantation embryo development. (A) hnRNPA2/B1 ICC image between control and knock-down 2-cell embryos. After 24H of microinjection of dsRNA, embryos were carried out immunostaining. Scale bars = 50 μM. (B) hnRNPA2/B1 protein intensity and (C) mRNA levels in the hnRNPA2/B1 dsRNA injected embryos were examined. (D, E) Developmental rates of porcine pre-implantation embryos injected with hnRNPA2/B1 dsRNA and control. (D) Representative images of HNRNPA2/B1 knock-down and control embryos at blastocyst stages after 144H microinjection. Scale bars = 500 μM. (E) Blastocyst rates was calculated of expanded and clear cavity formation blastocyst. Three experimental replicates were examined using 100-150 embryos per group. ***p < 0.01.

To confirm the developmental delay due to hnRNPA2/B1 knock-down, we confirmed the development from morula to blastocyst stage every 6H. Compared to control embryos, hnRNPA2/B1 knock-down embryos showed developmental delays (Fig. 3A and 3B). In blastocyst stage, m⁶A RNA methylation levels was significantly decreased in hnRNPA2/B1 knock-down blastocysts (Fig. 3C). Moreover, blastocyst size and total cell count in the blastocyst also significantly decreased in hnRNPA2/B1 knock-down embryos (Fig. 3D and 3E). These results demonstrated that hnRNPA2/B1 is important for porcine blastocyst formation.

Figure 3.Delay of blastocyst formation in knock-down of hnRNPA2/B1. (A) Bright field image after microinjection of 114H to 144H of control and hnRNPA2/B1 dsRNA. (B) Blastocyst rates changes between control and hnRNPA2/B1 dsRNA injection groups. (C) m⁶A RNA methylation levels was calculated from total RNA of blastocysts. (D) Immunostaining of hNRNPA2/B1 in blastocyst stage with DAPI. hnRNPA2/B1 was dis-localized within nucleus in hnRNPA2/B1 knock-down blastocyst. Scale bars = 50 μM. (E) Total cell number was calculated the DAPI positive cells in blastocyst stage. ***p < 0.01.

hnRNPA2/B1 knock-down alters global gene expression

Current research noticed that m⁶A regulator proteins induced the global gene expression changes in gamete cells (Zhao and He, 2017; Kwon et al., 2019). To investigate gene expression pattern alteration after hnRNPA2/B1 knock-down, we checked the transcription factor and cell junctional gene transcripts in blastocyst stage. The expression of pluripotency and cell polarity-related transcription factors, such as Pou5f1, SOX2, TRAP2C, CDX2, and Pard6B, significantly decreased in hnRNPA2/B1 knock-down embryos (Fig. 4). However, the expression of cell junctional protein ZO1, OCLN, and CDH1 were not significantly different. These results indicated that hnRNPA2/B1 knock-down changed the gene expression patterns during porcine early embryo development.

Figure 4.Effect of hnRNPA2/B1 knock-down on global gene expression patters. qRT-PCR analysis of pluripotency and cell junction-related gene expression in control and hnRNPA2/B1 knock-down embryos at blastocyst stage. Expression levels were normalized to that of GAPDH. Error bars indicate mean ± standard error of the mean (S.E.M). ***p < 0.01.

During the mammalian ZGA, new RNA is synthesized by epigenetic regulation and affect further early embryo development. Currently, m⁶A RNA methylation regulatory proteins including YTH and hnRNP domain family proteins can bind to mRNA and regulates pre-mRNA processing, such as mRNA stability, spicing, and export to nucleus to cytoplasm (Dreyfuss et al., 1993; Wang et al., 2014; Alarcón et al., 2015; Wang et al., 2015). These m⁶A RNA reader proteins contain multiple RNA recognition motifs (RRM) and have a high binding affinity to single stranded RNAs. These protein–m⁶A-methylated RNA complex performed dynamically RNA metabolism. Therefore, these m⁶A reader proteins might regulate mRNA biogenesis during mammalian early embryo development.

This study suggests that these m⁶A reader proteins widely affect mammalian development. YTHDF2, the m⁶A RNA reader protein belonging to YTH domain family, is involved in post-transcriptional maternal mRNA regulation and crucial for oocyte competence and zygotic embryo quality in mice (Ivanova et al., 2017). In chicken embryo development, m⁶A RNA methylation reader protein hnRNPA2/B1 is essential for differentiating smooth muscles through transcriptional activation of SMC gene expression and caused the embryonic defects (Wang et al., 2012). Moreover, we have previously revealed that hnRNPA2/B1 knock-down caused developmental delay and reduced ICM/TE ratio in mice (Kwon et al., 2019). Here, we report the hnRNPA2/B1 expression patterns during porcine early embryo development and show that hnRNPA2/B1 knock-down delays embryonic development and causes poor blastocyst quality and rates (Fig. 1-3). These results demonstrate that hnRNPA2/B1 has important developmental functions in porcine similar to those in other mammalian species.

hnRNPA2/B1 gene transcript expression pattern is highly upregulated at the 2-cell stage before ZGA (Fig. 1A). However, hnRNPA2/B1 transcript dramatically increases in mice at the 4-cell stage after ZGA, and then decreases after compaction. This different and temporal hnRNPA2/B1 expression between species may reflect species-specific functions related with further development such as blastocyst formation and ICM/TE transition during pre-implantation development, or species-specific lineage fate due to Oct4 and Cdx2 repression in the mammalian blastocyst. In hnRNPA2/B1 KD embryos, gene expression levels including transcription factor such as Pou5f1, Sox2, TFAP2C, CDX2 and PARD6B (Fig. 4). Previous our research in mouse paper (Kwon et al., 2019), transcript level of several transcription factor and ICM/TE cell fate determination genes was differ from controls. Moreover, m⁶A RNA methylation was also decreased in hnRNPA2/B1 KD blastocysts (Fig. 3C). These results indicate that KD of hnRNPA2/B1 caused the global gene expression regulation by affecting m⁶A RNA methylation levels. In summary, our results demonstrate that hnRNPA2/B1 is required for early embryo development, particularly pluripotent gene expression through m⁶A RNA methylation in mouse embryos. Future studies should determine the relationship between m⁶A demethylase like FTO and hnRNPA2/B1 during development. In addition, a study on interaction between OCT4 and HNRNPA2/B1 at chromatin remodeling level would be interesting and aid in understanding trophectoderm lineage establishment in mammalian embryos.

None.

Conceptualization, J-S.K., N-H.K., J.K., Y-J.J.; data curation, J.K., Y-J.J.; formal analysis, J.K., Y-J.J., S-B.Y., H.Y., C.Y.; investigation, J.K., Y-J.J., Y.K., J.L.; methodology, J.K., Y-J.J.; project administration, J.K., Y-J.J., N-H.K., J-S.K.; resources, J.K., Y-J.J., N-H.K., J-S.K.; supervision, N-H.K., J-S.K.; writing - original draft, J.K., Y-J.J., N-H.K., J-S.K.; writing - review & editing, J.K., Y-J.J., N-H.K., J-S.K.

This study was supported by grants from the KRIBB Research Initiative Program (KGM5162221).

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No potential conflict of interest relevant to this article was reported.