RESEARCH ARTICLE Molecular Reproduction & Development 81:820–832 (2014)

Exogenous Expression of OCT4 Facilitates Oocyte-Mediated Reprogramming in Cloned Porcine Embryos QIANQIAN JI, PEIQING CONG, HAIJING ZHAO, ZHENWEI SONG, GUANGYIN ZHAO, JINTAO GAO, YU NIE, AND YAOSHENG CHEN* State Key Laboratory of Biocontrol, Sun Yat-Sen University, Guangzhou, P. R. China

SUMMARY OCT4 is a well-established regulator of pluripotency and nuclear reprogramming. To determine if improving OCT4 abundance can facilitate oocyte-mediated reprogramming in cloned porcine embryos, we artificially increased OCT4 levels by coincubating donor cells with 50 ng/ml OCT4 plasmid. We observed higher rates of blastocyst formation (P < 0.05) and lower levels of blastocyst apoptosis in nucleartransfer-derived embryos carrying OCT4-incubated donor nuclei (OCT4-SCNT). The beneficial effect caused by exogenous expression of OCT4 involves epigenetic changes, wherein increased histone acetylation (AcH3K9) appeared in OCT4SCNT embryos at the one-cell and blastocyst stages and reduced histone methylation (H3K9me2) was observed at the one-cell stage (P < 0.05). There was a transient increase in exogenous OCT4 and an up-regulation of endogenous OCT4 level in OCT4-SCNT embryos (P < 0.05), while the expression pattern of epigenetic enzymes was changed. These modifications were accompanied by an up-regulation of CDX2, whose interaction with OCT4 is instrumental for implantation, and a downregulation of XIST, a negative indicator of reprogramming (P < 0.05). Taken together, our results support a role for exogenous expression of OCT4 in improving the efficiency of nuclear reprogramming while establishing a convenient and timesaving method to improve nuclear-transfer outcomes.

Mol. Reprod. Dev. 81: 820
832, 2014. ß 2014 Wiley Periodicals, Inc. Received 13 March 2014; Accepted 9 June 2014

INTRODUCTION Somatic-cell nuclear transfer (SCNT) is a very inefficient process, with only 1
3% of cloned blastocysts developing to term (Hochedlinger and Jaenisch, 2006). The main reason for this is incomplete or erroneous epigenetic reprogramming of the specialized somatic-cell nucleus, which results in aberrant embryonic gene expression (Zhao et al., 2010; Peat and Reik, 2012). On the other hand, the success of SCNT demonstrates that the nuclei of differentiated cells can be reprogrammed to an embryonic

ß 2014 WILEY PERIODICALS, INC.



Corresponding author: State Key Laboratory of Biocontrol School of Life Sciences Sun Yat-Sen University Guangzhou, P. R. China. E-mail: [email protected]

The authors have no conflict of interest to declare. Grant sponsor: The 973 Program; Grant number: 2010CB945404; Grant sponsor: The National Transgenic Major Program of China; Grant number: 2011ZX08006-005; Grant sponsor: China Agriculture Research System; Grant number: CASR-36

Published online 18 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22351

state by uncharacterized factors present in the ooplasm. As canonical transcription factors (e.g., OCT4, SOX2, KLF4,

Abbreviations: CDX2, caudal-type homeobox 2; eGFP, enhanced green fluorescent protein (ZsGreen); [Ac]H3K9[me2], [acetylated] histone 3 lysine 9 [dimethylated]; ICM, inner cell mass; OCT4 (POU5F1), POU domain class 5 transcription factor 1; PAR, parthenogenetic embryo; SCNT, somatic-cell nuclear transfer; XIST, X-inactive-specific transcript.

OCT4 FACILITATES OOCYTE MEDIATED REPROGRAMMING

and cMYC) are able to reprogram somatic cells to a pluripotent state, they may also improve cloning efficiency. The OCT4 (POU5F1) gene encodes a POU family transcription factor, and is expressed specifically in germ cells, the inner cell mass (ICM), and embryonic stem cells in the mouse. OCT4 encodes several alternatively spliced transcripts, which exhibit diverse expression patterns and functions. The OCT4A transcript variant (commonly known as OCT4) is responsible for the pluripotency properties of embryonic stem cells, whereas OCT4B is implicated in cell-stress responses (Wang and Dai, 2010). Generally, OCT4 is considered the most important reprogramming factor for the generation of induced pluripotent stem cells (Takahashi and Yamanaka, 2006; Yu et al., 2007; Kim et al., 2009). Due to its reprogramming capability, OCT4 is vital during the early development of cloned embryos. For example, OCT4 levels in the morula correlate positively with the development of cloned embryos (Cavaleri et al., 2008; Ji et al., 2013), while morpholino knockdown of OCT4 in mouse zygotes results in dose-dependent cleavage arrest (Foygel et al., 2008). Notably, OCT4 is also expressed in germ cells, demonstrating an up-regulation during oocyte maturation (Pesce et al., 1998). Nevertheless, the transcription level of OCT4 in mature oocytes varies significantly among individual oocytes (Monti and Redi, 2009; Pfeiffer et al., 2010), which may account for the high variability in their reprogramming capability. Together, these observations suggest that exogenous expression of OCT4 in oocytes may facilitate oocyte-mediated nuclear reprogramming, and hence improve the development of cloned embryos

although a recent report challenges this view by demonstrating that OCT4A-deficient oocytes are still able to reprogram fibroblasts into pluripotent cells (Wu et al., 2013). The efficiency of embryo gene-transfer methods varies greatly. In sperm-mediated gene transfer, the interaction between exogenous DNA and spermatozoa occurs only after a brief period of time (5 min) (Bevacqua et al., 2010). Similarly, short-term co-incubation of a transgene with somatic cells could be used to produce transgenic mammalian embryos by SCNT (Pereyra-Bonnet et al., 2011). Although the transgenic efficiency is relatively low, this latter method can still effectively introduce exogenous OCT4 into the oocytes. We therefore co-incubated donor cells with OCT4 plasmid prior to SCNT to determine if exogenous expression of OCT4 could facilitate oocytemediated nuclear reprogramming. The developmental capacity of the resultant OCT4-SCNT embryos was assessed using blastocyst formation rates, blastomere number, and cell apoptosis in blastocysts. Our results suggest that exogenous expression of OCT4 enhanced the development of porcine SCNT embryos. In an attempt to determine the mechanism by which exogenous OCT4 affects the development of SCNT embryos, we profiled epigenetic changes (AcH3K9 and H3K9me2) and the expression levels of several genes encoding epigenetic-modifying enzymes during the early development of OCT4-SCNT embryos. Together, these data implicated the involvement of epigenetic mechanisms.

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Further insight into the mechanism was obtained by analyzing the expression of other genes, including OCT4, X-chromosome inactive-specific transcript (XIST), and the trophectoderm-related caudal-type homeobox 2 (CDX2). The results of gene expression and epigenetic changes together indicate an improved reprogramming in OCT4SCNT embryos. Consequently, our data suggest that exogenous expression of OCT4 facilitates the development of SCNT embryos by improving nuclear reprogramming.

RESULTS Alignment of Human and Porcine OCT4 Gene Sequences The protein sequence, genomic organization, and chromosomal localization of the POU genes have been reported to be highly-conserved among species (Van Eijk et al., 1999). We also observed a high conservation in OCT4 sequence of 69.2% homology between pig (NM_001113060.1) and human (NM 002701.4) (Supplementary Fig. S1).

eGFP-Expressing Embryos Are Obtained in eGFP-SCNT and OCT4-SCNT Embryos In this study, eGFP signal was used as a reporter for the expression of exogenous genes. eGFP-positive blastocysts were observed in both eGFP-SCNT and OCT4SCNT groups on Day 7, albeit at a rate lower than 10% (Fig. 1), whereas control embryos did not exhibit any detectable green fluorescence. These results indicated that the exogenous genes introduced into the ooplasm were successfully expressed. As the intensity of eGFP was stronger in earlier-stage embryos than in blastocysts (Supplementary Fig. S2), the expression of these exogenous genes appears to be attenuated during later embryo development.

Developmental Rates to Blastocyst Are Higher in OCT4-SCNT Embryos To investigate the phenotype of exogenous OCT4 expression, we evaluated the efficiency of embryo development. In our experiments, the nuclear-maturation efficiency of oocytes was approximately 80%. Percentages of cleaved embryos, rates of blastocyst formation, and blastocyst cell numbers are shown in Table 1. Cleavage rates of the parthenogenetic group were significantly higher (PAR, 94.7%) than those of the other three groups (OCT4SCNT, 83.1%; eGFP-SCNT, 83.9%; SCNT, 86.3%), which is consistent with reports that parthenotes tend to exhibit a higher developmental potential than normal embryos during preimplantation development. Compared with SCNT and eGFP-SCNT embryos (19.5% and 19.0%, respectively), blastocyst formation rates were higher (P < 0.05) in OCT4-SCNT and PAR embryos (33.3% and 39.1%, respectively), with no significant difference between the former and the latter pairs. Among all groups, no difference in total cell numbers was observed.

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Figure 1. Detection of eGFP in blastocysts. Blastocysts were visualized under bright light (white) or with a blue light excitation (blue). Scale bar, 100 mm. SCNT, control blastocysts; eGFP-SCNT, with donor porcine embryonic fibroblasts co-incubated with eGFP plasmid (1, eGFP-positive; 2, eGFP-negative); OCT4-SCNT, with donor fibroblasts co-incubated with OCT4 plasmid.

When plasmid alone was injected into oocytes without fibroblasts, the efficiency of embryo development was significantly improved in the PAR-OCT4 group (with a partial removal of the cytoplasm) (48.9%) when compared with the PAR controls (29.0%) (Table 2). The PAR þ OCT4 (without removal of the cytoplasm) group, however, showed no difference from the PAR controls. There was no difference in cleavage rates or blastomere numbers among the groups, although PAR-OCT4, but not PAR þ OCT4, embryos had higher developmental rates to blastocysts than controls. With respect to these differences, we noticed that cytoplasm spilled from many PAR þ OCT4 oocytes after injection, and that these oocytes would undergo necrosis a few hours later. Thus, the injected volume itself likely caused some damage to PAR þ OCT4 oocytes, which negatively affected later embryo development. Taken together, these development results indicated that exogenous expression, rather than microinjection, of OCT4

leads to higher developmental rates of embryos to blastocysts.

Apoptotic Cell Number Is Lower in OCT4-SCNT Blastocysts Apoptosis is another metric used to evaluate blastocyst quality. The number of apoptotic cells per blastocyst was significantly lower in OCT4-SCNT and PAR than in control SCNTembryos (Fig. 2). There was no significant difference between the former group pairing.

Epigenetic Changes Are Involved in OCT4-SCNT Embryos Since epigenetic mechanisms can greatly contribute to the development of cloned embryos, we further analyzed changes of methylation and acetylation at histone H3 lysine

TABLE 1. Developmental Competence of Porcine Cloned Embryos Groups SCNTb eGFP-SCNTc Oct4-SCNTd PARe

No. of embryos cultrued

% of cleaved embryosa

% of blastocystsa

Cell no. per blastocysta

81 196 238 234

86.3  3.9a 83.9  1.2a 83.1  6.6a 94.7  3.2b

19.5  1.5a 19.0  4.1a 33.3  2.9b 39.1  5.9b

34.9  10.4 32.5  4.0 37.8  8.4 36.8  8.1

a,b: Values with different alphabets within columns are significantly different from each other (P < 0.05). a Mean  SE. Experiments were repeated four times. b Somatic cell nuclear transfer. c Donor cells were incubated with 50 ng/ml eGFP plasmid. d Donor cells were incubated in 50 ng/ml OCT4 plasmid. e Parthenogenetic embryos.

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TABLE 2. Developmental Competence of Porcine Parthenogenetic Embryos Groups PARb PARþOCT4c PAR-OCT4d

No. of embryos cultrued

% of cleaved embryosa

% of blastocystsa

Cell no. per blastocysta

143 123 93

93.0  1.0 91.8  4.1 90.2  3.8

29.0  6.4a 29.1  2.6a 48.9  9.3b

40.32  11.4 35.73  14.0 37.67  12.8

a,b: Values with different alphabets within columns are significantly different from each other (P < 0.05). a Mean  SE. Experiments were repeated three times. b Parthenogenetic embryos. c Parthenogenetic embryos injected OCT4 plasmid. d Parthenogenetic embryos injected OCT4 plasmid after removing equal amount of cytoplasm.

9 (H3K9), which are very important epigenetic modifications involved in epigenetic reprogramming after SCNT (Jackson et al., 2002; Fuks et al., 2003), by immunocytochemical analysis. We first determined the specificity of the primary antibody by staining embryos either without primary or secondary antibodies. The embryos showed no positive signals in these experiments, implying that the fluorescence signals we observed in this study were truly positive. Levels of acetylated H3K9 (AcH3K9) in one-cell stage embryos were significantly higher in OCT4-SCNT (P < 0.05) than in the SCNT and PAR groups (Fig. 3A1,B). In contrast, lower levels of dimethylated H3K9 (H3K9me2) were observed in OCT4-SCNT and PAR embryos compared with the SCNT group (P < 0.05) (Fig. 3A3,B). The

OCT4-SCNT embryos also showed a significantly higher abundance of OCT4 protein (Fig. 3A2,B). Together, these results suggested that the exogenous expression of OCT4 resulted in epigenetic changes in OCT4-SCNT embryos at the one-cell stage. By the blastocyst stage, levels of AcH3K9 were also higher in OCT4-SCNT embryos (P < 0.05) (Fig. 4), although no significant differences in H3K9me2 (Fig. 5) or OCT4 levels (data not shown) were observed among groups. Nonetheless, we noticed an interesting pattern of OCT4 localization: In both expanding and hatching OCT4-SCNT blastocysts, OCT4 was mainly localized to the ICM, with only trace amounts in the trophectoderm (Fig. 6).

Figure 2. Incidence of apoptosis in SCNT, OCT4-SCNT, and PAR blastocysts. A: Representative photographs of blastocysts stained by the TUNEL assay (green). Each sample was counterstained with DAPI to visualize DNA (blue). B: Number of apoptotic cells in each blastocyst. Values with different superscripts are significantly different (P < 0.05). n ¼ 16 (SCNT), 24 (OCT4-SCNT), and 24 (PAR).

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Figure 3. Immunocytochemical analysis of one-cell-stage embryos. A: Immunocytochemical analysis of H3K9 acetylation (A1) and dimethylation (A3), and OCT4 levels (A2) in SCNT, OCT4-SCNT, and PAR embryos. Each sample was counterstained with DAPI to visualize DNA. B: Quantification of AcH3K9/DNA, OCT4/DNA, and H3K9me2/DNA signal in SCNT, OCT4-SCNT, and PAR embryos. Values with different superscripts are significantly different (P < 0.05). The experiment was conducted three times, and at least 20 embryos were analyzed for each sample. Representative examples are shown.

Endogenous OCT4 Is Up-Regulated in OCT4-SCNT Embryos We then asked if the expression of endogenous OCT4 would be affected by the presence of expressed exogenous OCT4. To analyze the change in endogenous OCT4 expression after supplementation with exogenous OCT4, we applied quantitative reverse-transcriptase PCR using a combination of primers that were designed to discriminate between endogenous and exogenous OCT4

mRNA (Table 3). In this set-up, the forward primer for exogenous OCT4 was designed to correspond to the cytomegalovirus (CMV) promoter of the plasmid, and was used in conjunction with the same reverse primer to the OCT4 sequence. Thus, there should be no (or trace amounts of) exogenous OCT4 amplifications in the SCNT group due to an absence of corresponding sequence for the forward primer. Relative abundances of exogenous and endogenous OCT4 mRNA were analyzed

Figure 4. Immunocytochemical analysis of the acetylation state of H3K9 in blastocysts. A: Staining of AcH3K9 in SCNT, OCT4-SCNT, and PAR

blastocysts. B: Quantification of AcH3K9/DNA signal intensities in SCNT, OCT4-SCNT, and PAR blastocysts. Labeling intensity was expressed relative to that of the control embryos (set as 1). Values with different superscripts are significantly different (P < 0.05). The experiment was conducted three times and at least 10 embryos were analyzed for each sample. Representative examples are shown.

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Figure 5. Immunocytochemical analysis of the dimethylation state of H3K9 in blastocysts. A: Staining of H3K9me2 in SCNT, OCT4-SCNT, and PAR

blastocysts. B: Quantification of H3K9me2/DNA signal intensities in SCNT, OCT4-SCNT, and PAR blastocysts. Labeling intensity was expressed relative to that of the control embryos (set as 1). The experiment was conducted three times and at least 10 embryos were analyzed for each sample. Representative examples are shown.

in one-cell embryos (16 hr after activation) and blastocysts (Fig. 7). We observed significantly higher exogenous OCT4 levels in OCT4-SCNT embryos at the one-cell stage; how-

ever, this difference disappeared at the blastocyst stage (Fig. 7A), suggesting that the expression of exogenous OCT4 was transient. Surprisingly, levels of endogenous OCT4 mRNA were also higher in OCT4-SCNT than in SCNT embryos at both stages analyzed (P < 0.05) (Fig. 7B). Both OCT4-SCNT and PAR embryos had higher endogenous OCT4 levels than SCNT blastocysts (P < 0.05). These results indicate that exogenous expression of OCT4 during early stages improved the expression of endogenous OCT4.

Expression Patterns of Epigenetic Enzymes Are Modified in OCT4-SCNT Embryos

Figure 6. Immunocytochemical analysis of OCT4 in expanding and hatching OCT4-SCNT blastocyst. Each sample was counterstained with DAPI to visualize DNA. Representative examples are shown.

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OCT4 function is associated with multiple chromatinmodifying complexes and with multiple epigenetic pathways (Ding et al., 2012). Thus, we also analyzed the expression of genes encoding epigenetic-modifying enzymes, including those related to histone acetylation (HAT1 and HDAC1) and DNA methylation (DNMT1 and DNMT3a) (Fig. 8). Histone acetylation by histone acetylases (HATs) is thought to be associated with an active state of gene transcription. In our study, HAT1 expression was higher in OCT4-SCNT embryos at the one-cell stage (P < 0.05),

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TABLE 3. Primers Sequences Used for Real-Time RT-PCR Gene

Accession number

b-ACTIN

SSU07786

UBIQUITIN

M18159

DNMT1

NM_001032355

DNMT3a

NM_001097437

OCT4-plasmid (human OCT4) OCT4-30 UTR

NT_039353.8

HAT1

NM_003642

HDAC1

XM_005665200.1

CDX2

EU137688

XIST

KC149530

GFP plasmid

Primer sequence F: 50 -CTCGATCATGAAGTGCGACGT R: 50 -GTGATCTCCTTCTGCATCCTGTC F: 50 -TTCGTGAAGACCTTGACTG R: 50 -GGACTCCTTCTGGATGTTG F: 50 -TCGAACCAAAACGGCAGTAGT R: 50 -CGGTCAGTTTGTGTTGGAGAAG F: 50 -CTGAGAAGCCCAAGGTCAAG R: 50 -CAGCAGATGGTGCAGTAGGA CMV.F: 500 -TAGGCGTGTACGGTGGGAGG OCT4.R: 50 -GAGAAGGCGAAATCCGAAGC F: 50 -CAAACTGAGGTGCCCTTC R: 50 -ATTGAACTTCACCTTCCCTCCAACC F: 50 -TACAGCGGAAGATCCATCCAA R: 50 -CTGTTGTGCCTCTATCGCCA F: 50 -CCCCAGGGACTAGACAGGAA R: 50 -TGGAGAGGGATGGATGGTG F: 50 -AGAACCCCCAGGTCTCTGTCTT R: 50 -CAGTCCGAAACACTCCCTCACA F: 50 -CTTTGCCGCAGTGTTCCAGT R: 50 -GCCGCCATCTTTTGCTAT CMV.F: 50 -TGTACGGTGGGAGGTCTA GFP.R: 50 -AATAGACCGAGATAGGGTTGA

but was below the detection limit by the blastocyst stage (Fig. 8A). The expression of HDAC1, a major deacetylase in preimplantation embryos (Ma and Schultz, 2008), showed no significant difference in one-cell stage embryos among groups (Fig. 8B). For DNA methylation-related enzymes, the expression of both DNMT1 and DNMT3a were higher in OCT4-SCNT and SCNT than in PAR embryos (P < 0.05) at the one-cell stage, with no difference between the two SCNT groups (Fig. 8C,D). Surprisingly, DNMT3a was below the detection limit in OCT4-SCNT blastocysts, whereas it was detectable

Product size (bp)

Reference

114

Ju et al. (2010)

186

du Puy et al. (2011)

215

Ju et al. (2010)

238

Ju et al. (2010)

208 223

Wu et al. (2009)

139

Wu et al. (2009)

88 198

Li et al. (2011)

105

Li et al. (2011)

1,240

in SCNT and PAR groups (Fig. 8D). These results demonstrate that the expression patterns of genes encoding epigenetic-modifying enzymes were altered in OCT4SCNT embryos.

OCT4-SCNT Blastocysts Show Down-Regulation of XIST and Up-Regulation of CDX2 We further analyzed two more genes that are important for embryo development, XISTand CDX2. XIST is essential for triggering X-chromosome inactivation, and thus is

Figure 7. Expression levels of exogenous and endogenous OCT4 mRNA at the one-cell and blastocyst stage. A: Exogenous OCT4 mRNA and B: endogenous OCT4 mRNA in SCNT (black bars), OCT4-SCNT (gray bars), and PAR embryos (dark gray bars). Within a particular stage of development, bars with different superscripts (a, b, c) are significantly different (P < 0.05).

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Figure 8. Expression genes involved in reprogramming or development. A: Chromatin modification genes HAT1 and B: HDAC1, C: DNA

methylation genes DNMT1, and D: DNMT3a, E: X-chromosome inactivation-related gene XIST, and F: trophectoderm-related gene CDX2 between SCNT (black bars), OCT4-SCNT (gray bars), and PAR embryos (dark gray bars) at different stages of development. Within a particular stage of development, bars with different superscripts (a,b) are significantly different (P < 0.05).

important in early embryo development. CDX2, on the other hand, is tightly restricted in the trophectoderm lineage and is vital to cell-fate determination in early blastocysts (Beck et al., 1995). In this study, XIST transcription was below the detection limit in OCT4-SCNT blastocysts (Fig. 8E), whereas CDX2 was up-regulated (P < 0.05) compared with control embryos (Fig. 8F). These results indicated that the blastocyst quality of OCT4-SCNT is closer to that of normal embryos.

DISCUSSION To date, the oocyte is the only known cell in the adult body endowed with native reprogramming capabilities. When an oocyte fails to reprogram the somatic nucleus during SCNT, embryo loss and issues in later pregnancy become more probable. Nuclear reprogramming efficiency can be evaluated by determining the frequency of embryos that develop to the blastocyst stage and survive to birth after implantation, and by measuring the expression of pluripotent genes (Gomez et al., 2011). Short-term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos with 0.2% efficiency (Pereyra-Bonnet et al., 2011). By our method, PCR amplification was sufficient to detect the exogenous gene from DNA preparations taken after donor fibroblasts were incubated with plasmid (Supplementary Fig. S3). Indeed, we obtained eGFP-expressing embryos

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and blastocysts, implying that the incubation was sufficient to transfer some exogenous DNA into the embryos. The rate of eGFP-expressing blastocysts was lower than 10%, however, which suggests that expression from the plasmids was attenuated during development. The relative transcript level of exogenous OCT4 was higher in one-cell OCT4-SCNT embryos, but not in blastocysts, compared to SCNT or PAR controls (Fig. 7A). This transient expression of exogenous OCT4 is consistent with the fluorescent eGFP results, which showed an attenuating signal during embryo development (Supplementary Fig. S2), and may result from plasmid dilution during the rapid cleavages occurring over this period of embryo development. The transient effects of the exogenous OCT4 gene expression at the one-cell stage nevertheless appear to have a long-term influence on the embryo based on the up-regulation of endogenous OCT4 in embryos (Fig. 7B), which is consistent with the ability of OCT4 to activate its own expression through a positive autoregulatory loop (Okumura-Nakanishi et al., 2005). OCT4 is a well-known maternal-effect gene, and is an important contributor to the establishment of totipotency (Li et al., 2010; Wang and Dai, 2010). Although Wu et al. (2013) excluded a need for OCT4A in the establishment of totipotency, their data agree with the importance of OCT4 in the maintenance of totipotency. Thus, we predict that asyet-undefined factors are important for the establishment of totipotency, whereas OCT4A is primarily involved in its maintenance.

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A recent study by Foygel et al. (2008) suggested a role for OCT4 in the maternal-embryonic transition, which provides the first step towards the establishment of totipotency. This hypothesis supports the notion that OCT4 is a key contributor to the success of nuclear reprogramming. This role is further highlighted by the report that variations in OCT4 abundance influence the efficiency of somatic-cell cloning (Boiani et al., 2002; Bortvin et al., 2003). In mice, microinjection of OCT4 mRNA into the ooplasm results in higher blastocyst developmental rates of cloned embryos (Pfeiffer et al., 2010). Similarly, we observed increased blastocyst formation in cloned porcine embryos after co-incubation of donor cells with OCT4 plasmid (Table 1). Notably, the overexpression of exogenous OCT4 was transient in both studies, meaning that exogenous OCT4 is only necessary at initial stages of development. On the other hand, Goissis et al. (2012) reported no difference in the development rate when fibroblasts expressing OCT4 were used as donor cells in bovine SCNT. Further, ectopic expression of OCT4 can cause dysplasia in epithelial tissues in adult mice (Hochedlinger et al., 2005), suggesting that constant overexpression of OCT4 could interfere with normal development. It is worth noting that silencing of OCT4 in the trophectoderm is a prerequisite for the up-regulation of chorionic gonadotropin, which is secreted by the trophectoderm of peri-implantation blastocyst and is required for implantation and maintenance of pregnancy (Lanza et al., 2005). OCT4 expression is exclusive to the ICM in mouse embryos (Kirchhof et al., 2000) whereas it was found in both the ICM and trophectoderm of porcine blastocysts

albeit at lower levels in the trophectoderm (Kirchhof et al., 2000; Kuijk et al., 2008). Our results using porcine blascotysts are therefore in line with an enrichment of OCT4 in the ICM. Histone acetylation is associated with chromatin decondensation and gene activation, so a highly acetylated histones state would likely make a donor genome more amenable to reprogramming during SCNT. Therefore, the higher level of HAT1 expression in one-cell OCT4-SCNT embryos implies that these embryos can undergo more complete nuclear reprogramming (Fig. 8A). SCNT embryos, on the other hand, were reported to have higher levels of HDAC1; reduction of its activity by HDAC inhibitors such as trichostatin A or Scriptaid improves SCNTembryo survival (Iager et al., 2008; Whitworth et al., 2011). Accordingly, down-regulation of HDAC1 in one-cell OCT4-SCNTembryos (Fig. 8B) suggests that OCT4 operates on oocyte-mediated reprogramming, at least in part, through epigenetic mechanisms. Cloned embryos are also frequently plagued by high XIST expression levels, which are associated with incomplete reprogramming of the somatic nuclei. For example, increased XIST levels were observed in SCNT embryos in bovine (Goissis et al., 2012) and mouse (Nolen et al., 2005; Inoue et al., 2010), while knocking out XIST significantly improves term development of cloned mouse embryos (Inoue et al., 2010). We found the XIST levels in OCT4SCNT porcine blastocysts were below the detection limit (Fig. 8E), which may be an indicator of improved reprogramming.

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Mutant embryos lacking zygotic CDX2 fail to implant due to the abnormality of the trophectoderm-like cells (Strumpf et al., 2005). At the mRNA level, persistent expression of NANOG and OCT4 in the ICM coincides with an upregulation of CDX2 expression (Wu et al., 2013), which indicates an interaction between OCT4 and CDX2 gene regulation. In preimplantation embryos, CDX2 is initially coexpressed with OCT4, and under these conditions, they form a complex that can reciprocally repress their target genes in embryonic stem cells (Niwa et al., 2005). In blastocysts, this reciprocal interaction between OCT4 and CDX2 is important for further development and celltype differentiation. For example, the implantation of blastocysts and maintenance of pregnancy require the silencing of OCT4 in the trophectoderm (Liu et al., 1997), which is prerequisite for the expression of CDX2. Yet, an increase in CDX2 expression in SCNT embryos reconstructed with OCT4-overexpressing donor fibroblasts in bovine was reported (Goissis et al., 2012), and we observed a significant elevation in CDX2 expression in OCT4-SCNT blastocysts (Fig. 8F). Since CDX2 is downstream of lineage allocation (Ralston and Rossant, 2008), the observed up-regulation of CDX2 suggest a more normal process is occurring in these embryos. Taken together, the up-regulation of CDX2 resulting from exogenous expression of OCT4 may be a result of reciprocal repression, and will possibly contribute to implantation and further development. In conclusion, our results, combined with that of Pfeiffer et al. (2010), demonstrated that exogenous expression of OCT4 during the first cleavages can up-regulate endogenous OCT4 and facilitate oocyte mediated reprogramming. As compared to mRNA injection, we provide a more convenient and timesaving method to improve nuclear-transfer technology.

MATERIALS AND METHODS Chemicals and Reagents All chemicals for embryo culture and manipulation were purchased from Sigma
Aldrich, Inc. (St. Louis, MO), unless specified otherwise. Sterile plasticware was obtained from Nunclon (Roskilde, Denmark).

Collection and In Vitro Maturation of Porcine Oocytes The oocytes were collected and cultured for 44 hr, as described in our previous study (Ji et al., 2013). Ovaries were collected from prepubertal gilts at a local abattoir, and transported to the laboratory in 0.9% (w/v) NaCl at 30
358C within 2 hr. Ovarian follicles 2
6 mm in diameter were incised with a scalpel. Cumulus-oocyte complexes (COCs) were flushed out with Tyrode lactate-HEPES (TL-HEPES) containing 0.l% polyvinyl alcohol (PVA); those with uniform cytoplasm and at least three layers of cumulus cells were selected with a pipette and washed three times in TL-HEPES PVA. Oocytes were then washed in basic maturation medium (TCM-199 supplemented with 0.1% PVA,

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3.05 mM D-glucose, 0.91 mM sodium pyruvate, 26.19 nM sodium bicarbonate, 75 mg/ml penicillin G, and 50 mg/ml streptomycin) three times. Approximately 200 oocytes were transferred into 2 cm dishes containing 2 ml of preequilibrated maturation medium A (basic maturation medium supplemented with 10% porcine follicular fluid, 10 ng/ml of epidermal growth factor, 0.57 mM L-cysteine, 0.5 mg/ml luteinizing hormone, 0.5 mg/ml follicle-stimulating hormone). After 22 hr of maturation at 38.58C with 5% CO2 in air, oocytes were transferred into pre-equilibrated maturation medium B (basic maturation medium supplemented with 10% porcine follicular fluid and 10 ng/ml of epidermal growth factor), and cultured for another 22 hr.

Injection of Plasmid Suspension Alone, Without Fibroblasts The same volume used for the injection of fibroblasts incubated with plasmids (10 pl) was injected into oocytes to compare the influence of exogenous gene expression on PAR embryo development rates. Single oocytes were injected with approximately 10 pl plasmid suspension, with or without removing the same amount of cytoplasm (PAROCT4 or PAR þ OCT4, respectively). After a 2 hr equilibration in basic maturation medium in incubator, oocytes were activated with a single direct-current pulse, as above.

In Vitro Culture of Cloned Embryos Co-Incubation of Donor Cells With Exogenous DNA The nuclear-donor cells were porcine embryonic fibroblasts obtained from a Landrace fetus on Day 32 of pregnancy. Preparation of cell lines were carried out as previously described (Ji et al., 2013). The fibroblast suspension was centrifuged at 1,600 rpm for 5 min. The pellet was washed and diluted to 1.0  106 cells/ml with HEPES buffered TCM199 (30 mM NaCl, 0.595 mM NaHCO3, 0.3% bovine serum albumin, 0.1% HEPES, 50 mg/ml penicillin G, and 60 mg/ml streptomycin), and then kept for 1 hr at room temperature; most of the cells were round and highly refractile at the end of this period. After centrifuged and complete aspiration of the supernatant, 200 ml of incubation medium (2.8% sodium citrate with 100 mM EDTA, pH 7.4) was added and mixed well. Approximately 18.6 ml of the cell suspension and 1.4 ml of the constructed OCT4 plasmid (we cloned the human OCT4 genes Nm_002701.4 from the ATG to the stop codon, and inserted this coding sequence into pLVX-IRES-ZsGreen-CMV (Biowit Technologies) to achieve co-expression of OCT4 and eGFP; stock plasmid concentration was 714 ng/ml) were then mixed to make a 50 ng/ml plasmid concentration, and kept on ice for 5 min prior to SCNT. Porcine embryonic fibroblasts co-incubated with 50 ng/ml eGFP plasmid (pLVX-IRES-ZsGreen-CMV; the vector encodes an eGFP-flag ‘‘ZsGreen’’ marker that, if expressed, provides green fluorescence with a blue excitation) were used as a control.

Somatic-Cell Nuclear Transfer (SCNT) After incubation, 5 ml incubation medium including porcine embryonic fibrobalsts was immediately transferred into 15 ml HEPES-buffered TCM199 for nuclear transfer. A single round fibroblast and approximately 10 pl of surrounding medium was injected into the perivitelline space of enucleated oocyte. After a 2 hr equilibration in basic maturation medium in a culture incubator, the reconstructed couplets were fused and activated simultaneously with a single direct-current pulse of 120 kV/cm for 30 ms using a BTX Electro Cell Manipulator 2001 (BTX, San Diego, CA), in medium composed of 0.3 M mannitol, 1.0 mM calcium chloride dehydrate, 0.1 mM magnesium chloride hexahydrate, and 0.5 mM HEPES.

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After fusion, reconstructed embryos were transferred into four-well Nunc dishes containing 500 ml PZM-5, covered with mineral oil, and cultured at 38.58C, 5% CO2, and 100% humidity. The time of activation was defined as Day 0 (D0). Percentages of cleavage and blastocyst formation were evaluated separately on D2 and D7, respectively. The total cell number of blastocysts was counted on D7 by staining the embryos with 2.5 mg/ml 40 ,6-diamidino-2-phenylindole (DAPI) as follows: Blastocysts with intact pellucida were washed three times with PBS
PVA and fixed for 40 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4, freshly prepared) at room temperature. After washing in PBS
PVA, embryos were permeabilized in PBS with 0.5% TritonX-100 for 40 min at room temperature, and then stained with 2.5 mg/ml DAPI for 30 min in the dark at room temperature. After three washes in PBS
PVA, cell nuclei were quantified by fluorescence microscopy.

TUNEL Assay A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was carried out as described previous (Ji et al., 2013). Blastocysts were fixed and permeabilized as above, washed three times in PBS
PVA, and incubated at 38.58C in the dark for 30 min with TUNEL reaction medium (prepared immediately before use and kept on ice), covered with mineral oil. The reaction medium was composed of 2 ml enzyme solution, 18 ml label solution, and 20 ml PBS. In the negativecontrol group, the enzyme solution was replaced with 2 ml PBS. After three washes in PBS
PVA (10 min each time), blastocysts were stained with 2.5 mg/ml DAPI for 30 min in the dark at room temperature. After three washes in PBS
PVA, number of TUNEL-positive nuclei and total nuclei were quantified by fluorescence microscopy. Three separate experiments were performed.

Immunocytochemistry For immunofluorescence staining, embryos were stained as previous described, with slight modifications (Cong et al., 2013). Embryos were washed three times in PBS
PVA, fixed for 1 hr in 4% paraformaldehyde, and permeabilized with 0.5% TritonX-100 for 30 min at room

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temperature. After three washes in PBS
PVA, all samples were blocked overnight at 48C in 1% BSA and 0.05% Tween-20 in PBS (blocking solution). The samples were stained for 1 hr at room temperative with primary antibody (anti-acH3K9 or anti-dimethyl H3K9, diluted 1:200 [Upstate technology, Lake Placid, NY]; anti-OCT4 antibody, ab18976, diluted 1:200 [Abcam, Cambridge, UK]), followed by detection with anti-rabbit IgG secondary antibody (Alexa Fluor 555 [Cell Signaling Technology]). After three washes in PBS
PVA, the DNA was stained with DAPI in dark for 30 min. Samples were then washed in PBS
PVA, and mounted on slides with mounting medium for observation. Fluorescence intensity was quantified using Image-Pro Plus 6.0 software. Briefly, nuclear signal was outlined and mean fluorescence intensity was measured. The same encircled region was dragged to the cytoplasm of the same cell, and background fluorescence was measured. The specific signal was calculated by dividing nuclear values by cytoplasmic values. Experiments were repeated three times. For a given antibody, all images in a developmental series were taken at the same laser power.

Real-Time Reverse-Transcriptase PCR Quantification of all gene transcripts was performed by real-time reverse-transcriptase PCR using ACTIN and UBIQUITIN as internal standards. Primer sequences for all genes were derived from published data. The primer for the OCT4 plasmid was designed by Primer Premier 5.0. Sizes of amplified products are shown (Table 3). Total RNA was extracted from approximately 25 one-cell embryos or 10 blastocysts using the RNeasy Plus Micro Kit (Catalog no. 74034; Qiagen, Hilden, Germany), according to the manufacturer’s instruction. Reverse transcription was performed with the Reverse Transcription System (A 3500; Promega, Madison, WI) using Oligo (dT) 12
18 primer. Real-time PCR performed using SYBR1 Premier Dimer EraserTM (TaKaRa, Japan) with a LightCycler480 (Roche). The reaction mixture (total 10 ml) contained 5 ml master mix, 0.4 ml of each primer (10 mM), 1 ml cDNA template, and 3.2 ml distilled water. After denaturation at 958C for 10 sec, samples were subjected to 45 cycles amplification (958C for 15 sec, 608C for 30 sec, and 728C for 30 sec), followed by a melting cycle at 728C for 5 min and a step cycle starting at 658C with a 0.28C/sec transition rate to 958C. Three separate experiments were performed, each with four replicates. To calculate primer efficiency, PCR was performed from serial dilutions of cDNAs obtained as a template for each gene. The specificity of the real-time PCR product was confirmed by melting-curve analysis. The PCR product sizes were confirmed by agarose gel electrophoresis and staining with SYBR green 1 (Takara).

Statistical Analyses All sets of experimental data were analyzed by one-way ANOVA and LSD tests using SPSS 16.0 software (SPSS, Inc., Chicago, IL). Differences were considered significant

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at P < 0.05. Data were presented as mean  standard error of the mean.

ACKNOWLEDGMENTS This study was funded by the 973 Program (No. 2010CB945404), the National Transgenic Major Program of China (No. 2011ZX08006-005), and China Agriculture Research System (CASR-36).

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