m6a Methylation Inhibition by Cycloleucine Impaired Porcine Oocyte Meiosis and Early Embryonic Development via Remodeling Histone Modications and Altering Metabolism Related Gene Expression in Blastocysts

Oocytes maturation and early embryo development were regulated precisely by a series of factors at transcriptional and posttranslational levels. N6-methyladenosine (m 6 A) is the most prevalent modication in mRNA as a crucial regulator in RNA metabolism and gene regulation. However, the role of m 6 A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an inhibitor of m 6 A, could impair cumulus expansion, elevate mitochondrial reactive oxygen species (ROS) concentration and decreased oocytes maturation which partially caused by disturbed spindle organization and chromosomes alignment. Moreover, our results indicated that the CL treated parthenogenetic embryos arrested at 4-cell stage and showed worse blastocyst quality. CL treatment not only decreased the methylation levels of nucleic acid, H3K4me3 and H3K9me3, while increased the acetylation level of H4K16 during parthenogenetic embryos development in pigs. Furthermore, single cell RNA-seq (scRNA-seq) analysis indicated that CL treatment dramatically elevated the expression of metabolism-related (SLC16A1 and MAIG3 etc.) and maternal related (BTG4, WEE2 and BMP15 etc.) genes at blastocyst stage. gene


Introduction
The maturation of oocytes is a complex physiological and biochemical process [1]. In mammalian, oocyte maturation is accompanied by meiosis, which is precisely regulated by a series of factors and complex regulatory mechanisms [2,3]. The matured oocytes initiate early embryonic development once fertilization or parthenogenetic activation. Importantly, epigenetic modi cations have been found to play crucial roles during oocyte maturation and early embryonic development [4][5][6][7].
As the most common modi cation on mRNA, N6-methyladenosine (m 6 A) modi cation are dynamic and reversible in eukaryotes [8]. In mammals, m 6 A is regulated by methyltransferase complex (MTC), including methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14) and Wilms tumor 1associating protein (WTAP). An increasing number of studies indicated that m 6 A play crucial roles in disease development [9], stem cell fate determination [10] and early embryonic development [11,12]. As a major methyl donor in eukaryote, S-adenosylmethionine is synthesised by Methionine adenosyltransferase II (MAT) [13]. However, cycloleucine (1-aminocyclopentane-1-carboxylic acid, CL), a chemical inhibitor of methionine adenosyltransferase (MAT), could reduce the RNA m 6 A level [14]. Sun et al., suggested that CL treatment reduced the H3K4me3 level and resulted in embryos development arrest at morula stage in mice [15]. However, the underlying mechanism of CL treatment in regulating porcine embryo development remains largely unknown.
In the present study, we investigated the effect of different concentrations of CL on the porcine oocyte meiotic maturation and subsequent development after parthenogenetic activation (PA). Our nding indicated that CL treatment impaired cumulus expansion and caused aberrant spindle assembly in porcine oocytes. Furthermore, CL treatment altered m 6  The porcine ovaries were collected immediately after being slaughtered in a local abattoir, and then were transported to the laboratory within 2 hours in 0.9% NaCl supplemented with 5% penicillin-streptomycin at 35-36.5 °C. COCs (cumulus-oocyte complexes) were collected using a 10 ml syringe from 2-6 mm ovarian follicles. Then the COCs with more than three layers of cumulus cells were selected and cultured in the in vitro maturation (IVM) medium. A group of 200 COCs was cultured in 1 mL of maturation medium I (TCM-199 supplemented with 26 mM sodium bicarbonate, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.5 µg/mL follicle-stimulating hormone (FSH), 0.5 µg/ml luteinizing hormone (LH), 10 ng/ml epidermal growth factor (EGF), 0.1% PVA, 0.03% BSA and 0.1% penicillin/streptomycin) covered with liquid para n oil at 38.5 °C with 5% CO 2 for 22-24 h, and then cultured in maturation medium II (the same medium with medium I without hormone) until 42-44 h according to our previous description [16].
Oocytes at Metaphase I (MI) and Metaphase II (MII) stages were collected at 27 h and 42 h, respectively. For cumulus cell (CCs) treatment, CCs were isolated from COCs by 0.2% hyaluronidase and then were treated with CL at a working concentration of 20 mM.

Calculation of area of CCs expansion and viability
Cumulus expansion was measured during the COCs in vitro maturation period, as described previously [17]. Brie y, 40 COCs of each group were taken out of the incubators and captured by a uorescence microscope. The size of each COC within different groups were measured at 0 h, 24 h and 44 h of in vitro maturation. The ImageJ software was used to calculate the area of CCs expansion according with the description previously. The cumulus expansion rate of control group was calculated as 100% at 44 h of in vitro maturation, and it was performed at least three times. For the evaluation of cell viability, COCs were digested with 0.2% hyaluronidase [18]. After separating CCs with oocytes, the CCs were staining 0.4% trypan blue solution. Simultaneously, the cell viability was assessed by a light microscope (Nikon, Tokyo, Japan), living cells could not be stained with trypan blue. For oocytes survival rate, oocytes which possess intact zona pellucida and plasma membrane, evenly distributed cytoplasm were regarded as morphological survival. The cell viability was calculated as the percentage of viable cells out of total cells number and it was performed at least three independent replicates. Parthenogenetic activation (PA) of porcine oocytes Parthenogenetic activation of porcine oocytes was performed according with the description previously [19]. Brie y, after digestion with 0.2% hyaluronidase for 5 min, denuded MII oocytes (with the rst polar) were equilibrated in the activation medium (0.28 M mannitol, 0.5 mM Hepes, 0.1 mM MgCl 2 , and 1 mM CaCl 2 ) for almost 20 seconds. And then the oocytes were activated by an ECM2001 electro-fusion instrument (ECM2001, BTX, USA) with two direct-current pulses of 1.2 kV/cm for 30 µs. Then the oocytes were cultured in PZM-3 medium (108 mM NaCl, 10 mM KCl, 0.35 mM KH 2 PO 4 , 0.4 mM MgSO 4 .7H 2 O, 25.07 mM NaHCO 3 , 0.2 mM Na-pyruvate and 2 mM Ca-lactate.5H 2 0, 1 mM L-glutamine, 5 mM hypotaurine, 0.3% BSA, 20 mL/L BME amino acid and 10 mL/L MEM non-essential amino acid)) at 38.5 °C with 5% CO 2 for 7 days. The cleavage and blastocyst rates of embryos were visually checked at 48 h and 7 days after PA.
Immuno uorescence (IF) staining Immuno uorescence staining was performed according with our description previously [20]. Brie y, the oocytes or embryos were removed of zona pellucida with 0.5% acidic Tyrode solution. After washed with PBS/PVA (1 mg/1 mL) for three times, oocytes or embryos were xed with 4% paraformaldehyde (PFA) for 40 min at room temperature. The samples were permeabilized with 1% Triton-X 100/PBS for 20 min and blocked with 2% BSA/PBS for 1 h at RT. Then the samples were incubated overnight at 4 °C with primary antibodies according to manufacturer's recommended dilutions. After being washed with PBS/PVA for three times, samples were stained with secondary antibodies in the dark for 2 h at 37 °C. Then the DNA was stained with 10 µg/mL DAPI for 15 min. The oocytes or embryos were mounted on coverslips in ProLong Diamond Antifade Mountant reagent (Life Technologies, USA). A uorescence microscope (Nikon, Tokyo, Japan) was used to capture uorescent images by setting the same parameters for all groups. The uorescent images for α-tubulin were taken by a confocal laser-scanning microscope (LSM8800, Zeiss, Oberkochen, Germany). The relative uorescence intensity was analyzed by Image J software (National Institutes of Health, Bethesda, MD) according to the previous description.

Cell number counting of blastocyst
Total cell numbers of blastocysts were assessed and calculated by staining with Hoechst33342 (10 µg/mL). Brie y, blastocysts were xed with 4% PFA in PBS/PVA for 40 min, followed by permeabilization in 1% Triton-X 100/PBS for 30 min at RT. After being washed with PBS/PVA for 3 times

TUNEL assay
The embryos were removed of zona pellucida with 0.5% acidic Tyrode solution, and permeabilized by 1% Triton-X 100/PBS. After permeabilization, embryos were incubated with TUNEL solution from the In Situ Cell Death Detection Kit (Roche; Mannheim, Germany) in the dark at 37 °C for 1 h. Then, embryos were washed three times with PBS/PVA. DNA were stained with DAPI for 15 min. All samples were observed under a uorescence microscope (Nikon, Tokyo, Japan) after mounting. Each experiment was biologically replicated at least 5 times. The same exposure times and microscope settings were used for all captured images.

Reactive oxygen species (ROS) generation detection
The ROS level of oocytes were detected by a Reactive Oxygen Species Assay Kit (Beyotime Institute, Shanghai, China) following the manufacturer's instructions. Brie y, 100 denuded oocytes of each group were incubated with dichloro uorescein diacetate (DCFH-DA) probe (10 µmol/L) in the dark at 38.5 °C for The SMART-Seq2 technology was used to detect the global gene expression level in different groups. Brie y, the zona pellucida-free embryos (more than 20 blastomeres) were washed with PBS/PVA, and then were transferred into 0.2 mL tube with 4 µL lysis buffer (including 0.2 µL diluted ERCC spike-in) (1:1000). After SMART ampli cation, Ampure XP beads were used to purify the cDNA, and Qubit 3.0 Flurometer (Thermo Fisher Scienti c, Waltham, MA, USA) was used to measure the concentration of library, Agilent 2100 Bioanalyzer (Agilent Technologies) was used to detect the insert size and quality of ampli ed library. All libraries (PACL_4cell, PA_4cell, PACL_Bla and PA_Bla) were sequenced by Annoroad Biotechnology (Beijing, China) on an Illumina platform, and generated 150 bp paired-end reads. The clean reads were mapped to Sus_scrofa. Sscrofa11.1 and quanti ed gene expression levels as TPM (Transcripts Per Million).
Identifying differentially expressed genes (DEGs) and lncRNAs (DElncRNAs) DESeq2 was used for DEGs and DELncRNAs analysis of RNA-seq with a threshold of false discovery rate (FDR) < 0.05 and |log2FC| >1 between two groups.

Co-expression and Function analysis
The DEGs, target genes of DElncRNAs were performed function enrichment analysis, including GO and KEGG signaling pathway analysis using KOBAS 3.0 software. The lncRNAs-gene interaction networks were visualized with Cytoscape 3.4.0 (http://www.cytoscape.org/).

Predication of m 6 A sites in mRNA
The m 6 A sites of genes were predicated with the online software SRAMP (http://www.cuilab.cn/sramp) with default parameters. The very high/high/moderate con dence m 6 A sites were considered as the predicted m 6 A sites.

RNA isolation and real-time quantitative PCR (qRT-PCR)
Total RNA was isolated from porcine oocyte or embryos with RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. cDNA was synthesis by Transcript All-in-one First-Strand cDNA Kit (TransGen, Beijing, China), followed by qRT-PCR on a StepOnePlus™ Real-time PCR systems instrument (Applied Biosystems). The qPCR primers were designed by Primer3plus (http://primer3.sourceforge.net/webif.php) and synthesized by Comate biotech Co., Ltd. (Changchun, Jilin, China) (Table S1). In the present study, GAPDH was used as the internal control gene. The 2 −ΔΔCt method was used for the calculation of gene relative abundance.

Statistical analysis
All experiments were performed at least three independent replicates. GraphPad Prism8 statistical software (Graphpad Software, San Diego, CA, USA) was used for data analysis. Graphics were drawn using GraphPad Prism8 and R software (4.0). The results were presented as Mean ± SEM, and analyzed by t-test, one-way ANOVA test and multiple t test. p < 0.05 was considered statistically signi cant, p < 0.01 was considered extremely signi cant.

Results m 6 A methylation inhibition by CL treatment impaired oocyte quality and expansion of CCs via altering the m 6 A level
To investigate the effect of MAT inhibitor on porcine oocyte maturation, the COCs were cultured in different concentration of CL (10 mM, 20 mM and 40 mM) for 44 h at 38.5 °C and the survival rate and maturation rate were evaluated (Fig. 1a, and 1b). Our results showed that both the survival rate and maturation rate of oocyte in 10 mM, 20 mM, 40 mM CL group were signi cantly decreased (p < 0.01) (Fig. 1a, and 1b). The IF immunostaining analysis of m 6 A revealed that m 6 A was present both in the cytoplasm and nucleus (Fig. 1c). 20 mM CL treated oocytes displayed a markedly reduced m 6 A signal (p < 0.01), which was further con rmed by the uorescence intensity result of m 6 A (Fig. 1d).
In addition, we assessed the condition of cumulus cells (CCs) surrounding the oocytes during maturation (Fig. 2a). Results showed that the survival rates of CCs treated with CL (20 mM) were remarkably decreased at 24 h and 44 h of in vitro maturation compared with control group (p < 0.01) (Fig. 2b). Moreover, the expansion rate of CCs in the CL group was signi cantly lower than that of control group at 24 h and 44 h of in vitro maturation (p < 0.01) (Fig. 2c). Furthermore, the m 6 A level of CCs was detected by immunostaining and the m 6 A signal in CL treated-CCs was signi cantly decreased (Fig. 2d), which was consist with the result of uorescence intensity (p < 0.01) (Fig. 2e). Interestingly, our qRT-PCR assay results indicated that exposure to CL inhibited the expression of CCs expansion-related genes EFNB2, HAS2, PTX3 and antiapoptotic proteins BCL2 (p < 0.05) and promoted the expression levels of apoptosisrelated genes BAX and P53 (p < 0.01) ( Fig. 2f and 2 g). m 6 A methylation inhibition by CL treatment caused aberrant spindle assembly and oxidative stress in porcine oocytes Given the importance of spindle assembly in meiosis, next we sought to examine the spindle/chromosome structure in porcine oocytes after CL treatment. As showed in Fig. 3e, the control oocytes at MI stage showed a normal barrel-shape spindle with well-aligned chromosomes at the equatorial plate, while CL treated oocytes showed disorganized spindle and misaligned chromosome (Fig. 3a). The abnormal rates of spindle/chromosome structure were considerably higher in CL-treated oocytes than in the control group (Fig. 3b). Previous studies suggested that m 6 A was associated with oxidative stress which can impede oocyte maturation and embryonic development [21,22]. To investigate whether the CL would induce oxidative stress in oocyte, we next examined the relative ROS level in oocyte with DCFH-DA probes. Our result showed that the ROS level of CL-treated oocytes was signi cantly higher than that of control group (p < 0.01) (Fig. 3c), which was con rmed by the uorescence intensity result (p < 0.01) (Fig. 3d). m 6 A methylation inhibition by CL treatment decreased early developmental potency of parthenogenetic embryos To investigate whether the treatment of CL will exert a long-term effect on oocytes, we then analyzed the development ability of embryos produced by PA (Fig. 4a). The MII oocytes were selected and subjected to PA, and then the PA oocytes were cultured with CL (20 mM) exposure or control in PZM medium. Our results showed that the rates of cleavage and blastocyst were signi cantly decreased in CL group when compared with control group (p < 0.01) (Fig. 4b). Furthermore, the apoptosis analysis result suggested that the cell apoptotic signal (TUNEL) was considerably high in CL-treated blastocysts when compared with control group (Fig. 5a). Moreover, a massive accumulation of γH2A.X foci on the DNA were observed in CL-treated embryos (p < 0.01) (Fig. 5b) which was con rmed by uorescence intensity (p < 0.01) ( Fig. 5c and 5d). Subsequently, the blastocyst quality was assessed and our results indicated that the diameter and cell number of blastocysts were signi cantly reduced in CL-treated blastocysts (p < 0.01) ( Fig. 5g and 5 h). Furthermore, qRT-PCR result showed that CL treatment signi cantly decreased the mRNA levels of pluripotency genes NANOG and OCT4 at 2cell, 4cell and blastocyst (Bla) stage (p < 0.01) (Fig. 5i). Moreover, apoptotic-related genes BCL2 was signi cantly downregulated at stage of blastocyst (p < 0.05), while the mRNA level of anti-apoptosis gene BAX was signi cantly upregulated during embryonic development (p < 0.05) (Fig. 5i). m 6 A methylation inhibition by CL treatment reduced the RNA m 6 A methylation during PA embryogenesis Given that CL inhibits the catalytic of MAT2A and subsequently affects SAM level in eukaryotes, we then detected the mRNA expression level of methyltransferases (METTL3, METTL14 and WTAP) and nucleic acid methylation during embryogenesis by immuno uorescence staining. The qRT-PCR result showed that the transcript level of METTL3 was signi cantly increased at the 4-cell and 8-cell stage, while decreased at blastocyst stage compared with control group (Fig. 6a). The expression of methyltransferase METTL14 was signi cantly downregulated at 4-cell while upregulated at 8-cell and blastocyst stage (Fig. 6a). However, WTAP mRNA level was decreased at 2-cell, 4-cell and blastocyst stage (Fig. 6a). Interestingly, the m 6 A level was signi cantly decreased at 2-cell, 4-cell and blastocyst stage (p < 0.01) (Fig. 6b and 6c).

m 6 A methylation inhibition by CL treatment remodels histone modi cation during embryogenesis
Recently studies also reported that there is crosstalk between m 6 A and histone modi cations, which play vital role in embryo development [5,[23][24][25]. We then sought to investigate whether the CL treatment could lead to some change of histone modi cation during embryonic development which subsequently affect the embryo development. Firstly, we examined the methylation status of H3K9 and H3K4 by immune-staining after CL treatment during PA embryogenesis. The results indicated that the level of H3K9me3 was dramatically decreased in CL treated embryos at 4-cell and blastocyst stage as compared with control (p < 0.01) (Fig. 7a and 7b). Compared with control, the level of H3K4me3 was signi cantly decreased in CL treated embryos at 2-cell, 4-cell and blastocyst stage (p < 0.05) (Fig. 7c and 7d). In contrast, the level of H4K16ac was signi cantly upregulated at the 2-cell, 4-cell and blastocyst stage compared with control group (p < 0.05) (Fig. 7e and 7f). m 6 A methylation inhibition by CL treatment altered the global transcriptome of early embryos Previous study suggested that epigenetic modi cation has notable effects on gene expression [5,6,23]. To investigate the gene expression pro le of embryos after CL treatment, embryos treated with or without CL were collected at 4-cell and blastocyst stage and used for scRNA-seq (Fig. 8a). The correlation analysis between samples indicated that the difference between 4-cell and blastocyst was bigger than the difference between CL exposure and control (Fig. 8b). A total of 22, 682, 14352 and 13603 differently expressed (DE) genes between different groups (PACL_4cell-vs-PA_4cell, PACL_Bla-vs-PA_Bla, PACL_Blavs-PACL_4cell and PA_Bla-vs-PA_4cell) were identi ed in the present study (Fig. 8c), and the intersection of DE genes between different groups were shown in Venn diagram (Fig. 8d). In addition, we compared the global transcripts level between different groups. As illustrated in Fig. 9e, the global gene expression level was dramatically increased after CL treatment at the 4-cell and blastocyst stage of PA embryos (p < 0.001).
To exclude the effect of m 6 A on the DE genes expression, we then analyzed the potential m 6 A sites in the top twelve DE genes. The result showed that most of the DE genes have more than one moderatecon dence m 6 A site (Fig. 9a). Functional analysis result suggested that these DE genes mainly enriched on Protein processing in endoplasmic reticulum, P53 signalling pathway, Lysosome pathway and several metabolism-related pathways (Fig. 9b). Moreover, we also analyzed the DE lncRNAs in the present study. We got 28, 237, 6651 and 7215 DE lncRNAs between different groups (PACL_4cell-vs-PA_4cell, PACL_Blavs-PA_Bla, PACL_Bla-vs-PACL_4cell and PA_Bla-vs-PA_4cell), respectively. Co-expression analysis suggested that these DE lncRNAs were signi cantly correlated with DE genes at 4-cell and blastocyst stage of PA embryos. We nally constructed lncRNA-mRNA networks based on the integrated analysis of lncRNA/mRNAs co-expression and target gene prediction (Fig. 9f and 9 g). Noticeably, NONSUSTOO3873.1 and NONSUST017827.1 have the top degree of the network at 4-cell and blastocyst stage, respectively ( Fig. 9f and 9 g).

Discussion
Oocytes quality is closely related to fertilization and early embryonic development [26,27]. It is widely believed that high quality mature oocyte is usually characterized by some typical features, such as the well-organized CCs [28], high-proportioned oocyte survival rate [18] and rst polar body extrusion rate [29]. The communication between the oocyte and its surrounding CCs is essential for oocyte development and granulosa cell differentiation [30,31]. A growing number of studies indicated that m 6 A play crucial roles in the development of reproductive organ [32], gametogenesis [33], fertilization [34] and early embryonic development [7,35]. Cao et al. found that there exist abundant m 6 A modi cation sites in genes for porcine granulosa cells [36]. The differentially expressed m 6 A methylated genes were signi cantly enriched in several signaling pathways related with steroidogenesis, granulosa cell proliferation and follicular development [36]. Wang et al., reported that CL treatment impaired the maturation of porcine oocytes and reducing the RNA m 6 A level [37]. However, little is known about the effect of RNA m 6 A on the CCs expansion and ROS level of porcine oocytes. In our present study, we found that CL exposure signi cantly decreased the expansion of CCs and oocyte maturation via repressing the RNA m 6 A level. Meanwhile, the ROS level were dramatically increased in CL-treated oocytes. Taken together, CL reduced the m 6 A level, resulted in abnormal CCs expansion and ROS accumulation and spindle morphology and chromosome alignment.
Except for oocyte maturation, RNA m 6 A modi cation has been found to play notable effects on embryonic development [12,38]. In mice, CL exposure resulted in embryos arrested at morula stage [15]. In our study, CL treatment impaired the cleavage, blastocyst rates and total cell number of parthenogenetic embryos in pigs. We speculate that the endogenous m 6 A level might lead to different effects of CL on embryonic development in various mammals. Notably, Sun et al., found that CL reduced the H3K4me3 abundance in the mouse embryos treated with CL [15]. In the present study, we found that both the H3K4me3 and H3K9me3 levels were dramatically decreased in CL-treated medium during embryonic development in pigs. In addition, the level of H4K16ac was increased during parthenogenetic embryogenesis when treated with CL, indicating that crosstalk between m 6 A and histone modi cations during embryonic development. Moreover, the DNA damage signal (γH2A.X) was signi cantly increased at the blastocyst stage in CL-treated group, suggesting that DNA damage caused by m 6 A decrease might further impaired embryonic development.
To further clarify the potential genes regulated by m 6 A during embryonic development, we compared the global transcript levels at the stage of 4-cell and blastocyst. RNA-seq analysis suggested that CL treatment dramatically changed the transcript levels of large number of genes and lncRNAs at blastocyst stage, while only several DE genes and lncRNAs were identi ed at 4-cell stage. In mouse, Mendel et al., suggested that the downregulated SAM synthetase might trigger massive alteration in gene expression in the blastocysts [39]. We speculate that CL exposure may result in dysregulation of protein levels but not mRNA levels at 4-cell stage during parthenogenetic embryo development, which need to be further con rmed in the further. Notably, SLC16A1, MAGI3, BTG4, WEE2 and DNMT1 have more than ve potential m 6 A sites in their mRNA, indicating that they might be potential target genes affected by m 6 A during porcine early embryogenesis. The dysregulated expression of SLC16A1 and MAGI3, which can affect the pyruvate and lactic acid metabolism, may regulate metabolism homeostasis during early embryonic development [40][41][42][43]. Notably, several maternal mRNAs such as maternal-to-zygotic transition (MZT) licensing factor BTG4 [44,45], cell-cycle proteins WEE2 [46] and BMP15 [47] were also identi ed as DE genes in the present study, suggesting that the RNA m 6 A modi cation of these genes may regulate their expression thus participating embryonic development. In addition, little studies focus on m 6 Amediated lncRNAs during porcine embryonic development. Our result found the candidate lncRNAs NONSUST017827.1, NONSUST026375.1, NONSUST028825.1 and NONSUST021739.1 may target the metabolism-and embryonic development-related genes thus participating in embryonic development in pigs.

Conclusions
Taken together, our nding found that CL treatment could impair oocytes maturation and early embryonic development via reducing the nucleic acid methylation and histone modi cation levels. The reduced m 6 A methylation result in dysregulated expression of lncRNAs, metabolism-related (eg., SLC16A1 and MAGI3) and maternal mRNAs (eg., BTG4, WEE2 and BMP15) at blastocyst thus affecting early embryonic development (Fig. 10

Availability of data and materials
Sequencing data were deposited into NCBI Gene Expression Omnibus (SRA) database with accession numbers SRP289412.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.    The porcine parthenote development after CL treatment. (a) Representative images of parthenotes taken at 24 h, 48 h and 7 d. Scale bar, 100 μm. (b) The cleavage rates and blastocyst rates of parthenotes were recorded in control and CL-treated groups. Data are expressed as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01.