Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos

Theriogenology 132 (2019) 95e105

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Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos Qingqing Cai, Huiran Niu, Bingyue Zhang, Xuan Shi, Mengqin Liao, Zihao Chen, Delin Mo, Zuyong He, Yaosheng Chen, Peiqing Cong* State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 July 2018 Received in revised form 11 March 2019 Accepted 6 April 2019 Available online 14 April 2019

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis. © 2019 Elsevier Inc. All rights reserved.

Keywords: EZH2 Preimplantation development H3K27me3 Equilibrium Porcine

1. Introduction The development of early-stage mammal embryos is accompanied by changes in epigenetic modifications, such as DNA methylation, histone modifications, and X chromosome inactivation [1]. Since these changes occur rapidly during the first few cell divisions before the onset of transcription, factors stored in the oocyte (e.g. mRNA and proteins) are likely to induce them [2]. However, the function of many maternal epigenetic modifiers in early embryonic development remains unclear. Enhancer of zeste 2 (EZH2) is known as the catalytic subunit of Polycomb Repressive Complex 2 (PRC2) and functions as a histoneelysine N-methyltransferase of H3K27me3. The other two

* Corresponding author. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, PR China. E-mail address: [email protected] (P. Cong). https://doi.org/10.1016/j.theriogenology.2019.04.007 0093-691X/© 2019 Elsevier Inc. All rights reserved.

subunits of PRC2 are suppressor of zeste 12 homologue (SUZ12) and embryonic ectoderm development (EED) [3,4]. SUZ12 ensures the stability of EZH2 and facilitates nucleosome recognition [5,6], while EED is required to link EZH2 to histone H3 [7]. EZH2 interacts with EED via its domain II, which is conserved between Drosophila and mammals [8]. There is evidence in mice and human ES cells that PRC2 is able to repress various markers of cell differentiation [9,10], suggesting that PCR2 is implicated in regulating ESC differentiation. Especially, EZH2 has been shown to be critical for mouse ESC differentiation, as ESCs lacking Ezh2 show aberrant expression patterns of key differentiation regulators [11]. Together, these findings suggest that EZH2 is involved in regulating early embryogenesis and maintaining ESC identity, which is consistent with the observation that EZH2 is expressed during early embryonic development and in ESCs. EZH2 may achieve these roles by methylating H3K27 and regulating its target genes. Interestingly, there is research suggesting that the roles of EZH2 in histone methylation and

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maintenance of ESC identity can be partially complemented by EZH1, a homologue of EZH2. Unlike EZH2, EZH1 is preferentially expressed in differentiated adult tissues [12,13]. It has been shown in ESCs that EZH1 plays a role in determining the levels of H3K27me3 at a subset of PRC2 regulated genes. Depletion of Ezh1 in cells lacking Ezh2 resulted in the abolishment of residual methylation on H3K27 and the derepression of H3K27me3 target genes [11,14], suggesting that Ezh1 may help to ensure the effect of EZH2 on H3K27 methylation. However, EZH2 may also have independent functions that do not involve H3K27 methylation [15,16]. A recent study reported that the Ezh2 could directly regulate the epigenetic status of the NANOG promoter, suggesting a role for Ezh2 in regulating NANOG expression. This is consistent with the observation that the loss of Ezh2 in iPS cells is accompanied by increased levels of Nanog [17]. On the other hand, it has also been reported that Ezh2 can be negatively regulated in mouse preimplantation embryos by Oct4 and Sox2 at the post-translational level [18]. Since Oct4, Nanog and Sox2 are required for maintaining pluripotency in ESCs, these results suggest that EZH2 is critical to ensure the equilibrium between ESC self-renewal and differentiation. Given that the dynamics of early embryonic development also involves control of pluripotency and differentiation, which are primarily regulated by the maternal genome. However, little is known about the effect of maternal EZH2 on porcine early embryonic development. Considering the potential confounding influence of polyspermy, we aimed to investigate the expression patterns of EZH2 and its function during porcine preimplantation development using parthenogenetic embryos. 2. Materials and methods 2.1. Media and reagents All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise. All of the following solutions and media were filtered using a 0.2 mm filter. Oocyte in vitro maturation (IVM) medium consisted of TCM 199 supplemented with 0.1% (w/v) polyvinyl alcohol (PVA), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 26.19 nM sodium bicarbonate (NaHCO3), 75 mg/mL penicillin G, 50 mg/mL streptomycin, 0.57 mM L-cysteine, 0.5 mg/mL luteinizing hormone (LH), 0.5 mg/mL follicle-stimulating hormone (FSH), 10 ng/mL epidermal growth factor (EGF), and 10% porcine follicular fluid (PFF). Fusion medium contained 0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, 0.5 mM Hepes, pH 7.0 to 7.4. The embryo culture medium was porcine zygote medium 5 (PZM-5, PZM-3 [19] with a double dose of L-Glutamin). Oocyte manipulation medium consisted of 9.5 mg/mL TCM-199 powder, 0.595 mM NaHCO3, 0.75 mg/ mL Hepes, 50 mg/mL penicillin G, 60 mg/mL streptomycin, 30 mM NaCl and 3.0 mg/mL BSA at pH 7.2e7.4 and osmotic pressure 295e310 mOsm. Oocyte wash medium was Tyrode lactate-HEPES (114.0 mM NaCl, 3.2 mM KCl, 2.0 mM NaHCO3, 0.4 mM NaH2PO4, 10.0 mM sodium lactate, 2.0 mM CaCl2$2H2O, 0.5 mM MgCl2$6H2O, 10 mM HEPES, 0.25 mM sodium pyruvate, 2.186 mg/mL sorbitol, 65 mg/mL penicillin G and 25 mg/mL gentamycin sulfate) containing 0.0 l% (w/v) PVA (TL-HEPES-PVA) [20]. 2.2. Oocyte collection, oocyte IVM, parthenogenetic activation, and embryo in vitro culture Ovaries were collected from prepubertal gilts at a local abattoir, stored in saline, and transported to the laboratory at 30
Ce35
C within 2 h. Follicles between 3 and 6 mm in diameter were aspirated with an 18-gauge needle attached to a 10 mL syringe. Cumulus-oocyte complexes (COCs) within the follicular fluid were

allowed to settle by gravity at 38.5
C. Then they were flushed out with medium TL-HEPES-PVA. Only COCs with uniform cytoplasm and at least three layers of cumulus cells were selected with a pipette and washed three times in TL-HEPES-PVA. After washing three times with the maturation medium, approximately 200 oocytes were placed into 3 cm dishes, containing 2 mL preequilibrated IVM medium. After 22 h of maturation at 38.5
C with 5% CO2 in air, oocytes were transferred into IVM medium without cysteine and hormones for another 22 h culture. In vitromatured COCs were then denuded by vigorous pipetting in oocyte manipulation medium supplemented with 0.1% hyaluronidase. Oocytes with the extruded first polar body were used for in vitro development. Parthenogenetic activation was induced by a BTX Electro Cell Manipulator 2001 (BTX, San Diego, CA, USA) with a single DC pulse of 120 kV/cm for 30ms in the fusion medium. Then approximately 50 activated oocytes were cultured in each well of pre-equilibrated four-well Nunc dishes containing 500 mL PZM-5 covered with 300 mL mineral oil at 38.5
C and 5% CO2 in humidified air. Percentages of cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 h and 6e7 days after activation respectively. 2.3. siRNA construction and microinjection Three different siRNA species targeting EZH2 were designed with the online program BLOCK-iT RNAi Designer (http:// rnaidesigner.lifetechnologies.com/) following the manufacturer's instructions, and all the sequences of siRNA are listed in Supplemental Table S1. The siRNAs were diluted to 20 mM in water and stored at 80
C until use. Microinjection was performed in 20 mL oocyte manipulation medium with 7.5 mg/mL cytochalasin B on an inverted microscope (TE2000-U; Nikon, Japan) equipped with micromanipulation equipment (Nikon, Japan) at 38.5
C. Approximately 10 pL of EZH2 siRNA (20 mM) was microinjected into the cytoplasm of denuded MII stage oocytes. Oocytes with no injection or injected with scramble siRNA were used as control. The injected oocytes were activated according to the protocol after they were placed into pre-equilibrated IVM medium for 30 min at 38.5
C with 5% CO2 in humidified air. Then they were transferred to PZM-5 medium for culture. 2.4. Indirect immunofluorescence Oocytes and embryos used for immunofluorescence staining were washed twice in phosphate-buffered saline (PBS) containing 0.1% (w/v) bovine serum albumin (BSA), fixed for 40 min in 4% paraformaldehyde (PFA) in PBS, and permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature. After briefly washed in PBS containing 0.1% BSA, oocytes and embryos were blocked for 1 h at room temperature in PBS with 3% BSA. Then the samples were incubated overnight at 4
C with primary antibodies against trimethylated H3K27 (H3K27me3; 1:2000; Cat. No. 07e449; Milipore)/H3K4me3 (1:1000; Cat. No. ab8580; Abcam) and Anti-OCT4 (1:200; Cat. No. ab18976; Abcam) according to the manufacturer's protocol. After washing three times with PBS containing 0.1% BSA, secondary Chromeo 488 goat anti-rabbit immunoglobulin G (IgG) (Cat. No. 15041; Active Motif, Carlsbad, CA, USA) or Chromeo 546conjugated antibody (Cat. No. 15043; Active Motif, Carlsbad, CA, USA) was diluted 1:1000 and incubated for 1 h in the dark at room temperature. After three washes in PBS containing 0.1% BSA, the DNA was stained with 3 mg/ml 40 , 6-diamidino-2-phenylindole (DAPI; Sigma) in dark for 30 min. All samples were then washed and mounted on slides in 2% Vecta shield anti-bleaching solution (Vector Laboratories, Milpitas, CA, USA), which were analyzed by an inverted fluorescence microscope (ECLIPSE Ti-U; Nikon, Japan)

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equipped with a digital camera (DS-Ri2; Nikon, Japan) to detect the DAPI signals and immunofluorescence sequentially. Captured images were merged using Nikon NIS element software. In order to make relative comparisons, we kept constant settings for exposure and image capture, and all the images were assembled using Adobe Photoshop software (Adobe Systems, San Jose, CA, USA) with uniform adjustment of contrast or brightness to the whole images only when necessary. About 30 oocytes or embryos were processed for each treatment for evaluation, and the experiments were repeated three times with equivalent results. 2.5. Immunoblotting analysis A total of 200 porcine embryos or 50 porcine blastocysts per sample were lysed with 80 mL of protein lysis buffer with light shaking. Then, they were mixed with sodium dodecyl sulfate (SDS) buffer and boiled for 5 min at 100
C. Before analysis, the whole-cell lysates were stored at 80
C. Immunoblotting was based on procedures previously reported. Briefly, the protein samples were immediately cooled on ice and centrifuged at 12000 Хg for 5 min after being heated at 100
C for 3 min, which were then separated by SDS polyacrylamide gel electrophoresis from Bio-Rad PowerPace Basic (Bio-Rad, Hercules, CA, USA) at 80 V for 40 min and 120 V for 1 h with 5 mL of Multi-Coloured Standard markers loaded (Biyuntian, Shanghai, China). Then the samples were electrically transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ, USA). After blocking in TBST (TBS containing 0.5% Tween-20) containing 3% BSA at room temperature for 1 h, the membranes were incubated with anti-EZH2 (1:500; Cat. No. 21800-1-AP; Proteintech)/H3K4me3 (1:1000)/H3K27me3 (1:500) and anti-beta Actin (1:1000; Cat. No. ab8227; Abcam) antibody overnight at 4
C. After washing 4 times in TBST with 5 min for each washing, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; Cat. No. AP132P; Milipore). The membranes were finally extensively washed with TBST three times and then processed with the Kodak Image Station 4000 MM Pro detection system. 2.6. Quantitative real-time polymerase chain reaction (PCR) To examine the relative abundance of mRNA, 100e200 oocytes or 50e100 embryos of different stages were collected. Total RNA was extracted using RNAprep pure Micro Kit (Cat. No. DP420; TIANGEN, China) according to the manufacturer's instructions. The extracted RNA was quantified with spectrophotometry at 260/ 280 nm with a NanoDrop 2000 instrument (Thermo Scientific, USA), and then approximately 50 ng total RNA from each sample was taken for reverse transcription using the HiScript® II Q RT SuperMix for qPCR (þgDNA wiper) (Cat. No. R223-01; Vazyme, China). The synthesized cDNA was used for quantitative real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the most appropriate reference genes in early embryonic development of porcine, was used as an internal reference gene for normalizing the relative quantifications of target genes [21]. All the primers used for quantitative real-time PCR are listed in Supplemental Table S2. Quantitative real-time PCR was conducted according to manufacturer instructions of Hieff™ qPCR SYBR Green Master Mix (Yeasen, China) with LightCycler™480 (Roche). The PCR steps included incubations for 5 min 95
C, followed by 40 cycles of 95
C for 10 s, 60
C for 20 s and 72
C for 20 s. Dissociation curves were performed after each PCR run to ensure that a single PCR product had been amplified. Based on comparative threshold cycles (Ct) values, the relative quantification method was used to identify the expression level of mRNA. The transcript abundance of each

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gene was then calculated relative to that of the internal control gene. Then control group Ct values served as calibrators and were used subsequently to obtain DDCt values. The ratio change in the target genes relative to GAPDH or H2AFZ control gene was determined using the 2-DDCt method. Data are reported as means ± SEM for at least 3 replicates. 2.7. TdT-mediated dUTP nick end labeling (TUNEL) assay To assess the number of apoptotic cells in blastocysts, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were carried out with In Situ Cell Death Detection Kit (Cat. No. 11684795910; Roche, Mannheim, Germany) according to the manufacturer's instructions. Briefly, blastocysts were washed three times with 0.1% BSA in PBS, and fixed for 40 min in 4% paraformaldehyde, followed by permeabilizing in PBS with 0.5% Triton X-100 for 40 min at room temperature. After washing three times in PBS containing 0.1% BSA, blastocysts were incubated for 30 min in TUNEL solution (an immediately mixture of 2 mL Enzyme Solution, 18 mL Label Solution, and 20 mL PBS) at 38.5
C in the dark. Positive control blastocysts were incubated in DNase I for 20 min at 37
C before performing TUNEL. In the negative control group, the Enzyme Solution was replaced with 2 mL PBS. After being washed three times in PBS containing 0.1% BSA, blastocysts were stained with 3 mg/mL DAPI for 30 min in the dark at room temperature. At last, all the samples were washed and aspirated into 2% Vecta shield on microcapillary glass slides for observation. The total number of cells per blastocyst was calculated by counting DAPI-stained nuclei. For each blastocyst, apoptotic indices were calculated by the number of apoptotic cells against the total number of cells. The experiments were repeated three times with equivalent results. 2.8. Statistical analysis The data are shown as mean ± SEM of results from at least three independent experiments. All the experiments were repeated at least three times. Differences in relative expression assayed by quantitative PCR and embryo nuclear number as well as apoptotic resistance were tested for significance by Student t-test. Differences in cleavage rate and blastocyst rate were analyzed by ANOVA after arcsine square root transformation. Differences at P < 0.05 were considered statistically significant. 3. Results 3.1. Dynamic changes of EZH2 mRNA abundance during porcine oocyte maturation and preimplantation development are accompanied by changes in H3K27me3 levels There are two mammalian homologues of Enhancer of Zeste: EZH1 and EZH2. To determine which one is crucial for the trimethylation of H3K27 in porcine early embryonic development, the abundances of EZH1 and EZH2 in oocyte and parthenogenetic preimplantation embryos were examined by qPCR. During oocyte maturation, both EZH1 and EZH2 displayed relatively high levels of expression. EZH1 remained relatively stable after GVBD, while EZH2 kept decreasing until the MII stage, when it showed a similar level to that in GV (Fig. 1A and B). Interestingly, as shown in Fig. 1A, the RTPCR results indicated that the expression of EZH1 had a moderate increase in its abundance from the PN to 4-cell stage before reaching a peak at the 8-cell stage. Then it decreased until the blastocyst stage. By contrast, the expression level of EZH2 increased immediately after parthenogenetic activation, but it then decreased during early cleavage stages, reaching the lowest level at the morula stage, after which it increased anew at the blastocyst stage (Fig. 1B).

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Fig. 1. Dynamic expression pattern of polycomb subunits and H3K27me3 in porcine oocytes and parthenogenetic embryo development. Transcriptional profile of EZH1 (A) and EZH2 (B) using quantitative real-time PCR. Oocytes at germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI), metaphase II (MII) stages and embryos at the putative zygote (PN), 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages were collected. The levels of the transcripts were normalized against GAPDH. GV oocytes were used as calibrator sample (expression set to 1). Data are presented as the mean ± SEM; **P < 0.01. (C) Immunofluorescence staining using anti-H3K27me3 antibodies in porcine oocytes and early embryos at different stage. Original magnification was  200 for the oocytes and embryos.

Immunostaining of H3K27me3 in oocytes and embryos of different development stages was performed. H3K27me3 was easily detectable at all stages of oocytes and PN. The latter is spontaneously formed after parthenogenetic activation. However, starting at the 2-cell stage, it was not detectable from the 4-cell to 8-cell stages before increasing at the blastocyst stage (Fig. 1C). Therefore, it is the dynamic expression of EZH2, but not EZH1, that is concomitant with trimethylation of H3K27 during the early development of porcine parthenogenetic embryos. 3.2. EZH2 is required for early porcine parthenogenetic embryo development To examine if EZH2 is conserved among mammals, the protein sequences of EZH2 from pig (NM_001244309.1), human (NM_001203249.1), mouse (NM_001146689.1), rat (NM_001134979.1), cattle (NM_001193024.1), sheep (XM_012177324.2) and rabbit (XM_008258065.2) were compared. The porcine EZH2 showed 92.81% and 97.3% homology to that of human and mouse, respectively. Furthermore, the homology of all sequences was above 90%, demonstrating that EZH2 is highly conserved among these species (Fig. 2A). To detect the effect of EZH2 on the development of porcine parthenotes, we did gene knockdown using short interfering RNA

(siRNA) in parthenogenetic embryos (Fig. 2B). EZH2 siRNA1 was most effective when EZH2 siRNA1/2/3 duplexes were transfected into porcine fetal fibroblast cells (Supplemental Fig. S1A). After the completion of IVM, siRNA duplexes were microinjected into MII porcine oocytes to knock down EZH2. The average efficiency of microinjection was 81.5% as measured by injection of the BLOCK-iT Alexa Fluor 555 control siRNA (Invitrogen, Germany) (Supplemental Fig. S1B). Non-injection and nonspecific siRNA injection groups were used as negative controls. As shown in Table 1, the nonspecific siRNA had no obvious influence on either the viability or the developmental potential of embryos (Table 1). After parthenogenetic activation, the efficiency of EZH2 knockdown was assessed by RT-PCR, which showed that the expression of EZH2 significantly decreased as early as 12 h after siRNA injection, and reached the lowest level on Day 4 (73.79% ± 0.01, P < 0.001, Fig. 3A). Western blot analysis of Day 4 embryos also consolidated the success of EZH2 knockdown (Fig. 3 B. Day 0 means the day of parthenogenetic activation). The developmental competence of parthenogenetic embryos were evaluated by cleavage and blastocyst rates. The cleavage rate showed no difference among groups. However, the blastocyst rate of the EZH2-siRNA group (32.9 ± 3.4%) was decreased compared to the control group (47.4 ± 2.0%) and the Nonspecific siRNA group (49.0 ± 7.3%, P < 0.01). Furthermore, a reduced total cell number

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Fig. 2. Experimental design and conservation of EZH2 across species. (A) Protein sequence of EZH2 from porcine, human and other common mammals were aligned using DNAman software. (B) Overview of parthenogenetic activated embryos and developmental stages in porcine.

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Table 1 Development of porcine parthenogenetic embryos after injection with EZH2 siRNA in the MII oocytes. Treatment

No. of replications

No. of oocytes

Rate that cleaved (%±SEM)

Rate of blastocysts (%±SEM)

No injection Nonspecific siRNA EZH2-siRNA

6 4 6

969 675 1089

(68.6 ± 0.6)a (67.5 ± 1.0)a (70.2 ± 1.0)a

(47.4 ± 2.0)a (49.0 ± 7.3)a (32.9 ± 3.4)b

No. of total cells (mean ± SEM) (56.1 ± 1.9)a (53.6 ± 2.3)a (47.1 ± 1.8)b

Values in the same column with different letters (a, b) differ significantly (P < 0.05).

(Table 1) and an increased apoptotic index (fragmented cell number/total cell number) of blastocysts (11.4 ± 1.7 vs. 6.1 ± 0.6 and 5.9 ± 1.1, P < 0.01, Fig. 3C and D) were observed in the EZH2-siRNA group by performing the TUNEL assay. 3.3. Knockdown of EZH2 restrains trimethylation of H3K27 on day 4 and disturbs the balance of H3K27me3-H3K4me3 at the blastocyst stage Many reports support that EZH2 is responsible for the catalytic formation of H3K27me3 during embryonic development [3,4], and

that there are bivalent domains of H3K4me3 and H3K27me3 in embryonic stem cells [9,22,23]. To understand the effect of EZH2 knockdown during porcine embryogenesis, 4th-day parthenogenetic embryos and blastocysts from the EZH2-knockdown group and the control group were collected for examination of H3K27me3 and H3K4me3. Western blot analysis showed lower abundance of H3K27me3 but higher level of H3K4me3 on Day 4 (Fig. 4A). The expression level of KDM5B, which is the demethylase of H3K4me3, was significantly decreased, while genes encoding the corresponding methyltransferases, KMT2A, KMT2B, KMT2C and KMT2D were almost unchanged in Day 4 embryos. Meanwhile, the

Fig. 3. RNA interference technology reveals that knockdown of EZH2 can promote the apoptosis of cells in blastocysts. (A) Quantitative real-time PCR analysis the abundance of EZH2 at 12 h, 24 h, 48 h, 72 h and 96 h after EZH2-siRNA injection and parthenogenetic development, respectively. The levels of the transcripts were normalized against GAPDH; data are presented as the mean ± SEM; **P < 0.01. (B) Western blot analysis the abundance of EZH2 at 96 h after EZH2-siRNA injection. Each sample was normalized to b-Actin content. (C) Staining for apoptotic cells in porcine blastocysts (using TUNEL); positive control-induced apoptosis by DNaseI and negative control-processed without Enzyme Solution. Scale bar ¼ 100 mm. (D) Percent of apoptotic cells in blastocysts. 60 embryos of each group were detected. Data are presented as the mean ± SEM; **P < 0.01.

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Fig. 4. Knockdown of EZH2 disturbs the bivalent modification balance of H3K27me3- H3K4me3 in porcine embryos. (A) Western blot analysis of H3K27me3 and H3K4me3 abundance after knockdown of EZH2. 200 embryos per sample were collected at 96 h after injection EZH2-siRNA and parthenogenetic development. Each sample was normalized tob-actin. (B) Quantitative real-time PCR analysis of other members of PRC2 (EED, SUZ12), EZH1 and H3K4me3 demethylase KDM5B in day 4 embryos after EZH2-knockdown. The levels of the transcripts were normalized against GAPDH. Data are presented as the mean ± SEM; **P < 0.01. (C) Quantitative real-time PCR analysis of H3K4me3 methyltransferases in day 4 embryos after EZH2-knockdown. (D) Quantitative real-time PCR analysis of PRC2 (EZH2, EED, SUZ12) and H3K4me3 demethylase KDM5B in the blastocysts from the control and EZH2 knockdown groups. **P < 0.01. (E) Western blot analysis of the abundance of H3K27me3 in the blastocysts from the control and EZH2 knockdown groups. 50 blastocysts per sample were collected. (F) Immunofluorescence staining of the H3K4me3 and H3K27me3 in the blastocysts from the control (n ¼ 76) and EZH2-knockdown groups (n ¼ 87). H3K27me3 (green) was probed with rabbit anti-H3K27me3 antibodies (1:2000) and detected by using Chromeo 488-conjugated antibodies (1:1000). H3K4me3 (red) was probed with rabbit anti-H3K4me3 antibodies (1:1000) and detected by using Chromeo 546-conjugated goat anti-rabbit antibodies (1:1000). Scale bar ¼ 100 mm. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

expression of EZH1 was down-regulated, whereas no noticeable decrease was detected in the other two members of PRC2, EED and SUZ12 (Fig. 4B and C). In addition, the expression of KDM5B and EED was significantly downregulated at the blastocyst stage as a result of EZH2 knockdown (Fig. 4D, P < 0.01). Consistently, Western blot analysis displayed a decrease of H3K27me3 after EZH2 knockdown (Fig. 4E). An increase in the level of H3K4me3 was detected after

EZH2 knockdown in blastocysts, though the antibody against H3K4me3 that we used for Western blot analysis was not specific enough to make great quality images (Supplemental Fig. S1). The immunofluorescence further confirmed the correlation between the decrease of H3K27me3 and the accumulation of H3K4me3 (Fig. 4F).

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3.4. Knockdown of EZH2 induces apoptosis at the blastocyst stage Depletion of Ezh2 has been shown to promote spermatogonial differentiation and apoptosis [24]. What's more, upregulation of EZH2 has been found in various types of solid tumors, and recent studies have demonstrated that EZH2 can suppress apoptosis in a variety of cancers [25e27]. To determine if EZH2 is implicated in regulating apoptosis so as to affect the quality of blastocysts, the expression of genes related to apoptosis were examined by RT-PCR. The results showed that BCL-XL (an anti-apoptotic gene) and BAD (a pro-apoptotic gene) were both decreased after the knockdown of EZH2 (Fig. 5A, P < 0.01). However, the ratio of BCL-XL to BAX, which represents the capacity of apoptosis resistance [28], decreased in the EZH2-siRNA treatment groups compared to the control group. This is consistent with the result of the TUNEL assay (Figs. 3C and 5B, P < 0.01). 3.5. Knockdown of EZH2 influences the expression level of pluripotency genes and TETs In addition to trimethylation of histone H3K27, EZH2 has also been reported to play a role in the regulation of other genes, such as

transcription factors, Oct4, Nanog, Sox2, and TETs [17,18,29]. RT-PCR and immunofluorescence assay were therefore performed to examine the expression of OCT4, NANOG and SOX2 to further investigate the causes behind the developmental abnormalities in EZH2 knockdown blastocysts. After EZH2 knockdown, the expression of all these three transcription factors was down-regulated, with more significant decreases in OCT4 and NANOG (Fig. 5C, P < 0.01), which was confirmed furthermore by the immunofluorescence of OCT4 (Fig. 5D, P < 0.01). Interestingly, in the EZH2siRNA group, the abundances of TET2 and TET3 were greatly elevated while the expression of TET1 was reduced (Fig. 5E, P < 0.05). Moreover, we observed a significant upregulation of a portion of differentiation-related genes (Fig. 5F, P < 0.05). 4. Discussion The roles of EZH2 during oocyte maturation, early embryogenesis, and somatic cell nucleus transfer (SCNT) have been studied extensively in mouse [12,30]. How it functions during porcine preimplantation development, especially the maternal effect, however, remains poorly understood. Using siRNA interference, we demonstrated that EZH2 knockdown disrupted the equilibrium

Fig. 5. EZH2 knockdown affects the potential of parthenogenetically activated blastocysts. (A) Quantitative real-time PCR analysis of apoptosis-related genes in the blastocyst from the control and EZH2 knockdown groups. The levels of the transcripts were normalized against GAPDH. Data are presented as the mean ± SEM; **P < 0.01. (B) The ratio of expression level of BCL-XL/BAX in the blastocyst from the control and EZH2 knockdown groups. Data are presented as the mean ± SEM; **P < 0.01. (C) Quantitative real-time PCR analysis of pluripotent genes in the blastocyst from the control and EZH2 knockdown groups. **P < 0.01. (D) Immunofluorescence staining of OCT4 using anti-OCT4 antibodies at blastocyst from the control (n ¼ 30) and EZH2 knockdown groups (n ¼ 30). Scale bar ¼ 100 mm. (E) Quantitative real-time PCR analysis of TET family genes in the blastocyst from the control and EZH2 knockdown groups. *P < 0.05. (F) Quantitative real-time PCR analysis of differentiation-related marker genes in the blastocyst from the control and EZH2 knockdown groups. *P < 0.05.

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between differentiation and pluripotency of cells in the pig parthenogenetic embryo, suggesting that EZH2 is integral to its developmental program. During pig oocyte maturation and preimplantation development, EZH1 and EZH2 both underwent dynamic changes. However, only that of EZH2 was accompanied by changes in global H3K27me3 levels (Fig. 1), suggesting a correlation between EZH2 and H3K27me3. Despite this correlation, different results have been obtained regarding the dynamics of both EZH2 and H3K27me3. In our study, H3K27me3 was persistently detectable during the whole process of oocyte maturation and at the PN stage but barely detectable in cleavage stage embryos (from 2-cell to compacted morula stage). However, at the blastocyst stage, our results showed that H3K27me3 became detectable again, which is consistent with that reported by Gao et al. [31]. But there is also a study reporting the missing of H3K27me3 staining at the blastocyst stage in porcine [32]. A few reasons may contribute to the apparent differences in the staining patterns of H3K27me3 in porcine blastocysts. Firstly, it may be due to the difference in the compared developmental stages and the different time points when the embryos were collected. Both Gao et al. and Park et al. have performed H3k27me3 staining with in vitro fertilization (IVF) embryos. The later found that the staining was from faint to absent in blastocysts, while the former performed staining on hatched blastocysts, which develop further than blastocysts, and showed that H3K27me3 was higher in the trophectoderm than in the epiblast. Because of the lower ratio of ICM cells/total cells in parthenogenetic blastocysts than that in IVF embryos [33], this might make it relatively easier for us to detect H3K27me3 in parthenogenetic embryos. In particular, there are also inconsistent reports regarding the staining patterns of the H3K27me3 mark in the blastocysts of murine. Wu and Zhang et al. found that the intensity of H3K27me3 staining in blastocysts was weaker than that in morula [18,34]. Erhardt et al. observed a weak overall staining in trophectoderm cells except in some special areas, whereas the ICM showed an intense staining [35]. Interestingly, Yang et al. showed that H3K27me3 modification distributed with high levels in both the ICM and the trophoblast cells in early blastocysts, and the distribution was more extensive than that in morula. Also, they found that the H3K27me3 mainly aggregated in the ICM of hatched blastocysts [36]. Secondly, there may be differences in the sensitivity of the experimental methods used. The global level of H3K27me3 in pig blastocysts might just below the detectable level by some methods. We speculated that H3K27me3 would increase by at least the blastocyst stage since it plays a crucial role in X-chromosome inactivation. Furthermore, our results about the H3K27me3 staining in porcine blastocysts are similar to the observations of Huang et al. [37]. Besides, the abundance of H3K27me3 in parthenogenetic blastocysts has also been detected by Western blot in our study (Fig. 4E). Thirdly, these discrepancies might originate from different antibody accessibility. Trimethylated H3K27 was surrounded by higher-order chromatin structures, which have the potential to disturb the process of antibody accessing the antigen, especially when different antibodies were used in these reports. Similarly, different results have also been reported for the expression pattern of EZH2 during porcine preimplantation development. For example, Gao et al. showed a steady increase of EZH2 from the 4-cell to the hatched blastocyst stage, but Park et al. showed a reduction in EZH2 expression during the same period of embryonic development. This difference is probably due to differences in the origin of embryos, the culture conditions, the sample processing and the reference genes. Interestingly, the activity of Ezh2 in mouse is also controversial. Some reported that it was already apparent in late stage zygotes [38,39], but Wu et al. showed that Ezh2 expression peaked during the zygote stage but then

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gradually decreased from the 2-cell stage and reached the lowest level at the blastocyst stage [18]. These findings above indicate that the establishment and maintenance of global H3K27me3 requires EZH2, which is regulated by a potential mechanism in different mammals before implantation. Here we showed that microinjection of siRNA at the MII stage efficiently reduced endogenous EZH2 mRNA and protein. Although the percentage of cleaving embryos was not affected, blastocyst formation and blastocyst cell number were both reduced in EZH2 knockdown groups (Table 1). Similar results have been reported in mouse after loss of Ezh2 at the pronuclear stage [40]. Ezh2deficient blastocysts have compromised potential for outgrowth, preventing the establishment of ES cell and trophoblast cell lineages [41]. Furthermore, the mutation or maternal depletion of Ezh2 in mouse embryos results in failures in post-implantation development [12,35]. The above studies support the notion that EZH2 plays an important role in embryonic development. EZH2 is a lysine methyltransferase for H3K27me3. Our results showed that EZH2 knockdown moderately decreased the levels of H3K27me3 at the 4-cell and the blastocyst stages. In contrast, it temperately increased the levels of H3K4me3 and reduced the abundance of H3K4me3 histone demethylase KDM5B at both 4-cell and blastocyst stages. Previous research has identified bivalent modifications of H3K27me3 and H3K4me3 in KDM5B-depleted porcine embryos and ESCs [23,37]. The bivalent domain is associated with genes poised for activation during embryonic development, e.g. early differentiation-related genes GATA3, GATA6 and HAND1, which are all increased as a result of EZH2 knockdown (Fig. 5F) [42]. Gata3 is a trophoblast-specific gene that is associated with suppression of pluripotency [43,44]. There is evidence that ectopic expression of PRC2 in mTSCs induces deposition of the H3K27me3 mark at the Gata3 locus and abrogates its transcription. Furthermore, embryos with depleted Kdm5b or ectopic expression of Eed contain abrogated expression of Gata3 in the TE lineage, resulting in them either failing to develop to the blastocyst, or failing to implant [45,46]. HAND1 has been reported to be transcriptionally repressed by PRC2 through bivalent chromatin modifications in both human and murine ES cells [10,23]. The expression of HAND1 was increased in EZH2-deficient porcine parthenotes but not significantly. This is similar to that in mouse embryos, indicating that there are other alternate transcription factors affecting cell fate together [40]. In the present study, the abundance of three pivotal stemness genes, OCT4, SOX2 and NANOG, were also affected at the blastocysts stage after EZH2 knockdown. Consistently, inhibition or knockdown of Ezh2 impairs the reprogramming process in cloned mouse embryos, ESCs, or iPSCs, mainly because of aberrant expression of development-related genes and unscheduled differentiation resulting from the lack of PRC2-dependent H3K27me3 [10,17,47]. A previous study found that Ezh2 co-localizes with developmentrelated genes in the ICM of mouse blastocysts [34]. Wu et al. reported that the promoter activity of Ezh2 was suppressed by either Oct4 or Sox2 in NIH/3T3 cells in a dose-dependent manner. They also showed that Oct4 and Sox2 were negative regulators of Ezh2, primarily at the post-translational level [18]. On the other hand, Ezh2 could modulate the expression of Sox2 during reprogramming by influencing H3K27me3 on its enhancer region [48]. Moreover, Ezh2 has been shown to contribute to the equilibrium of Nanog-high and Nanog-low states in ES/iPS cells through modifying H3K27me3 at the promoter of NANOG [17]. Taken together, these reports suggest a close correlation between these pluripotency regulators and Ezh2 in mouse embryonic development and cellular reprograming. This may explain the impaired development of embryos to blastocysts in the EZH2-siRNA group. Accordingly, the crucial mechanism that enables EZH2 to support embryonic

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development may also be involved in adjusting pluripotencyassociated transcription factors. Our results showed that knockdown of EZH2 enhanced the expression of apoptosis-related genes, such as BCL-XL, BAX, CASPASE3 and BAD, and accelerated cell apoptosis in blastocysts, which is harmful to embryonic development. This has nothing to do with the microinjection process since there was no significant difference between the nonspecific-siRNA microinjection group and the noninjection control group in levels of apoptosis. The ratio of BCL-XL to BAX reflects cell's capacity to resist apoptosis: the former inhibits apoptosis by blocking cytochrome c, while the latter promotes apoptosis by stimulating the release of cytochrome c for activation of caspases [28,49]. In our study, although BCL-XL and BAX were both down-regulated in EZH2 knockdown parthenotes, the ratio of BCL-XL to BAX also decreased, suggesting a compromised antiapoptotic potential of cells in these embryos. Similar findings have been reported in spermatogonial stem cells [24], prostate cancer stem cells [27], as well as tumors [50,51], all of which show an induction of apoptosis by EZH2 deficiency. EZH2 can also interact with Ten-Eleven Translocation gene family members (TET1, TET2, and TET3), which convert 5methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), to cause changes in patterns of histone methylation and DNA methylation [52]. Huang et al. found that the expression levels of all the TETs members were increased in porcine blastocysts after knockdown of KDM5B, a demethylase of H3K4me3 [37]. Intriguingly, we observed that the production of TET2 and TET3 was increased while that of TET1 was remarkably down-regulated after EZH2 knockdown, indicating that each of the three may have its own particular function besides converting 5 mC into 5hmC. Studies in porcine preimplantation embryos have identified that the conversion of 5 mC to 5- 5hmC is dynamic during embryo development, and is mainly regulated by TET3 at the initial stage and by TET1 at the later stage. Notably, the expression of NANOG was significantly decreased after TET3 knockdown at the zygote stage [53]. It is also documented that TET1 co-localizes with PRC2, and EZH2 has been identified as a co-factor of TET1 in controlling DNA methylation [52,54]. Furthermore, Tet1 is required for ES cell maintenance and preferentially binds to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes [29]. Knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation and the downregulation of Nanog due to the methylation of the Nanog promoter [55]. This is consistent with our observation that knockdown of EZH2 resulted in the downregulation of TET1 and pluripotency factors such as NANOG and the increase of GATA3, a marker of trophoderm. This may also contribute to the impaired competency of embryonic development. 5. Conclusion We presented data that the expression of EZH2 is stage specific and is consistent with the dynamic change of H3K27me3 during preimplantation parthenogenetic development. Depletion of EZH2 caused a lower developmental capacity and a decreased quality of blastocysts, which may result from the disturbed bivalent balance of H3K27me3 and H3K4me3 in the EZH2 knockdown embryos. The downregulation of NANOG and the upregulation of GATA3 might be induced indirectly, given that the expression of TET1 is decreased. Together with changes in the expression of pluripotency related genes OCT4 and SOX2, differentiation related genes and apoptosisassociated genes, these findings are reasonable, in a way, to propose that EZH2 interacts with different complexes to get involved in the mechanisms underlying cell self-renewal, differentiation initiation and apoptosis in pig early parthenogenetic embryos.

Author contributions Qingqing Cai, Peiqing Cong, Delin Mo and Yaosheng Chen conceived, designed and directed the study. Qingqing Cai and Huiran Niu performed the experiments. Bingyue Zhang, Xuan Shi and Mengqin Liao interpreted the data. Qingqing Cai and Zihao Chen wrote the manuscript. Peiqing Cong and Zuyong He revised the manuscript. All authors have read and approved the manuscript for publication. Declaration of interest None of the authors have any conflicts of interest to declare. Role of the funding source Funding sources were not involved in research design, collection, analysis and interpretation of data, writing of the report, and decision to submit the article for publication. Acknowledgments We thank Yueting Zhang for her assistance in collecting ovaries and selecting cumulus-oocyte complexes. This work was supported by National Transgenic Major Program (2016ZX08006003-006). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.theriogenology.2019.04.007. References [1] Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, et al. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci U S A 2001;98: 13734e8. [2] Solter D, Hiiragi T, Evsikov AV, Moyer J, De Vries WN, Peaston AE, et al. Epigenetic mechanisms in early mammalian development. Cold Spring Harbor Symp Quant Biol 2004;69:11e7. [3] Cao R, Zhang Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol Cell 2004;15:57e67. [4] Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, Reinberg D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev 2002;16: 2893e905. [5] Pasini D, Bracken AP, Jensen MR, Lazzerini DE, Helin K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. EMBO J 2004;23:4061e71. [6] Montgomery ND, Yee D, Chen A, Kalantry S, Chamberlain SJ, Otte AP, et al. The murine polycomb group protein Eed is required for global histone H3 lysine27 methylation. Curr Biol 2005;15:942e7. [7] Tie F, Stratton CA, Kurzhals RL, Harte PJ. The N terminus of Drosophila ESC binds directly to histone H3 and is required for E(Z)-dependent trimethylation of H3 lysine 27. Mol Cell Biol 2007;27:2014e26. [8] Kuzmichev A, Jenuwein T, Tempst P, Reinberg D. Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3. Mol Cell 2004;14:183e93. [9] Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, et al. Chromatin signatures of pluripotent cell lines. Nat Cell Biol 2006;8:532e8. [10] Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006;441:349e53. [11] Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 2008;32:491e502. [12] O'Carroll D, Erhardt S, Pagani M, Barton SC, Surani MA, Jenuwein T. The Polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol 2001;21:4330e6. [13] Ezhkova E, Lien WH, Stokes N, Pasolli HA, Silva JM, Fuchs E. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev 2011;25:485e98. guelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, et al. EZH2 is [14] Be required for germinal center formation and somatic EZH2 mutations promote

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