Discovery of porcine maternal factors related to nuclear reprogramming and early embryo development by proteomic analysis

Differentiated cell nuclei can be reprogrammed to a pluripotent state in several ways, including incubation with oocyte extracts, transfer into enucleated oocytes, and induced pluripotent stem cell technology. Nuclear transfer-mediated reprogramming has been proven to be the most efficient method. Maternal factors stored in oocytes have critical roles on nuclear reprogramming and early embryo development, but remain elusive. In this study, we showed most of porcine oocytes became nuclear matured at 33 h of IVM and the rate had no significant difference with oocytes at 42 h of IVM (p > 0.05). Moreover, the cleavage and blastocyst rates of SCNT and PA embryos derived from 42O were significantly higher than that of 33O (p < 0.05). But 33O could sustain IVF embryo development with higher cleavage and blastocyst rates comparing to 42O (p < 0.05). To clarify the development potential difference between 33O and 42O, 18 differentially expressed proteins were identified by proteomic analysis, and randomly selected proteins were confirmed by Western blot. Bioinformatic analysis of these proteins revealed that 33O highly synthesized proteins related to fertilization, and 42O was rich in nuclear reprogramming factors. These results present a unique insight into maternal factors related to nuclear reprogramming and early embryo development.


Introduction
Reprogramming of sperm or somatic cells into pluripotency has great attraction and value in both research and therapy. Recent advances in iPS have clearly indicated that differentiated cells can acquire pluripotency by overexpressing a small number of transcript factors, such as Oct4, Sox2, Klf4, and c-myc [1]. However, the reprogramming process is slow (at least 1 to 2wk), and the efficiency is low (typically about 1 %) [2]. In contrast, reprogramming by oocytes occurs within one or two cell cycles, and often in a majority of embryos, suggesting oocyte-mediated reprogramming resets differentiated cell states more efficiently than the iPS method and more factors must participate in the process [3][4][5]. So, understanding of these oocyte factors will provide us important information on nuclear reprogramming and embryo development.
Given how little is known about the factors in mammalian oocyte, there are several recent reports utilizing proteomics approaches to explore the oocyte proteomes in cattle, pig and mouse [6][7][8][9][10][11][12][13][14][15]. For example, Calvert et al. identified 8 highly abundant HSPs and related chaperones in the mature mouse egg by two-dimensional electrophoresis [16]. Vitale et al. used 2DE and MS to identify 12 proteins that appeared to be differentially expressed between GV and MII murine oocytes [12]. However, it has some limitations in identifying proteins that are related to nuclear reprogramming and early embryo development. So, a comparison of the proteomes of oocytes with different abilities of nuclear reprogramming and development may aid in the identification of the maternal factors.
In the study, we checked the effect of oocyte maturation time on early development of IVF, PA and SCNT embryos. Moreover, mass spectrometry was applied to identify the maternal factors related to nuclear reprogramming and early embryo development, and we successfully discovered 18 differentially expressed proteins between 33O and 42O in pig.

IVM of porcine oocytes
Porcine ovaries were collected from a local slaughter house and kept in saline at 32-37°C. Antral follicles (3-5 mm indiameter) were aspirated with an 18-gauge needle. Aspirated oocytes with an evenly granulated cytoplasm and at least three uniform layers of compact cumulus cells were selected and cultured in four-well plates (Nunc, Naperville, IL, USA) containing 500 μL of maturation medium that was TCM199 (Gibco) based medium plus 0.05 μg/ml EGF, 0.5 μg/ml LH and FSH, at 39°C in 5 % CO 2 in air [17]. The rates of polarbody extrusion were calculated from 16 h-42 h of IVM. Matured porcine oocyte was obtained at 33 h and 42 h for further experiments.

IVF and cloned embryos production and in vitro culture
For IVF, freshly ejaculated sperm-rich fractions were collected from fertile boars, and following a short incubation at 38.5°C, the semen was resuspended and washed three times in Dulbecco's Phosphate Buffered Saline (DPBS) supplemented with 0.1 % (w/v) bovine serum albumin (BSA) by centrifugation at 1500 × g for 4 min. The spermatozoa concentration was measured using a hemocytometer, and the proportion of motile sperm was determined. The spermatozoa were diluted with modified Tris-buffered medium (mTBM) to an optimal concentration. Cumulus-free oocytes at 33 h or 42 h of IVM were washed three times in mTBM. Approximately 30 oocytes were inseminated in 50-ml drops of mTBM at a final sperm concentration of 3 × 10 5 /ml for 6 h.
After 12 h co-incubation with spermatozoa, oocytes were fixed for 30 min in 4 % paraformaldehyde, and permeabilized for 20 min in 0.1 % triton X 100 following wash in PBS. Then oocytes were stained with 10 μg/ml Hoechst 33342 to visualize the nuclei. Samples were observed and photographed by using a Nikon Eclipse 80i epifluorescence microscope (Nikon, Tokyo, Japan). Oocytes with one or more swollen sperm head(s) and/or male pronuclei were considered as fertilization and fertilization rate was evaluated.
The procedure for porcine SCNT has been described previously [18]. After 33 h or 42 h of IVM, the oocytes were treated with 1 mg/ml hyaluronidase (H3506, Sigma-Aldrich, Missouri, USA) to remove the surrounding cumulus cells. Oocytes with a clearly extruded first polar body were selected as recipient cytoplasts. Cumulus-free oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm with a glass pipette 25 mm in diameter in TCM199-Hepes plus 0.3 % BSA and 7.5 mg/ml Cytochalasin B. Porcine ear fetal fibroblasts at passage 5 served as donor cells and were injected into the perivitelline space of enucleated oocytes. Injected oocytes were placed in fusion/activation medium (0.3 M mannitol, 1.0 mM CaCl 2 , 0.1 mM MgCl 2 , and 0.5 mM HEPES). Fusion/ activation was induced with 2 DC pulses of 1.2kv/cm for 30 msec on a BTX Elector-Cell Manipulator 2001 (BTX, San Diego, CA). Cumulus-free oocytes were directly activated by the same parameters as SCNT procedure for producing PA embryos.
The embryos were cultured in porcine zygote medium-3 (PZM-3) at 39°C in 5 % CO 2 in air. The cleavage and blastocyst rates were assessed at 48 and 156 h after activation, and the number of blastocyst cells was examined by nuclear staining with 5 μg/ml Hoechst 33342.

Oocyte collection and proteomic analysis
Zona pellucida of more than 10,000 33O and 42O were removed (Additional file 1: Figure S1) and total proteins were extracted using ultrasonic wave and lysis buffer. The lysis buffer consisted of 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 65 mM DTT, 2 % (v/v), and 1 % (v/v) protease inhibitor cocktail. The protein concentration was determined by the Bradford method and pH was adjusted to 8.5 with 50 mM NaOH. In fluorescent 2D DIGE, protein from 33O and 42O were equally pooled together and labeled with Cy2 as internal standard, and the two samples were labeled with Cy3 or Cy5, separately. Labelled samples were mixed in the rehydratation buffer before to be loaded onto the into 24-cm pH 3-10 IPG strips (Bio-Rad) and run in a single two-dimensional gel. Then, the gels were scanned using the Typhoon 9410 scaner with excitation/emission wavelengths of 488/ 520 nm for Cy2, 532/580 nm for Cy3 and 633/670 nm for Cy5. Image analysis were performed by Decyder software suite 5.02 (GE Healthcare) which allow the comparison of the different combination corresponding to the experimental conditions. An independent t-test was used to determine the significance between the experimental groups. P-values less than 0.05, and fold-changes greater than 1.5 were considered statistically significant.
For protein identification, the spots of interest were cut from the gels, and washed with 25 mM NH 4 HCO 3 and 50 % ACN solution and dehydrated with 100 % ACN sequentially and dried by centrifugal lyophilization. The gels were digested with 15-20uL of 0.01ug/ul trypsin (Promega) in 25 mM ammonium bicarbonate for 15 h at 37°C. The supernatants were collected, and the tryptic peptides were extracted from the gel sequentially with 5 % TFA at 40u for 1 h and with 2.5 % TFA, 50 % ACN at 30°C for 1 h. The extracts were pooled and completely dried by centrifugal lyophilization. Digested peptides with matrix (50 % acetonitrile, 0.1 % TFA containing 3 mg/ml alpha-cyano-4-hydroxy cinnamic acid matrix) were then spotted on the target plate. Samples were analyzed by MALDI-TOF-TOF MS (4800 Proteomics Analyzer, Applied Biosystems) in positive reflectron modeat fixed laser fluency with low mass gate and delayed extraction. Database searching was carried out using Mascot version 2.2 (MatrixScience, London, UK) via GPS explorer software (ABI) version 3.6 combining MS and MS/MS interrogations on NCBI pig proteins database. In the searching parameter, modification was set as carbamido-methylation, oxidation, and a maximum of one missed trypsin cleavage was permitted. Tolerance of precursor and fragment ions were both set to 0.2 Da. The reported proteins were always those with the highest number of peptide matches. All identified proteins were those with a statistical significance (p ≤ 0.05) and the best ion score.

Quantitative realtime PCR
Total RNA was extracted from at least 100 oocytes using the PureLink TM Micro-to-Midi system (Invitrogen) according to the manufacturer's instructions, and reverse transcription was used to generate cDNAs using the Pri-meScript TM RT Reagent Kit (TaKaRa). Real-time PCR was performed using SYBR Premix Ex Taq TM (TaKaRa) and the 7500 Real-Time PCR System (Applied Biosystems). The reaction parameters were 95°C for 30 sec, followed by 40 two-step cycles of 95°C for 5 sec and 60°C for 34 sec. Primers for each gene were listed (Additional file 1: Table  S1). 18S rRNA was used as a reference gene. C t values were calculated using Sequence Detection System software (Applied Biosystems), and the amount of target sequence normalized to the reference sequence was calculated as 2 -△△Ct .

Western blot
Oocytes removed zona pellucida, stored at −80°C, were transferred to 10 μl cold 40 mM sodium phosphate, pH 7.6, containing 50 mM NaCl, 50 μM sodium orthovanadate, 10 mM sodium fluoride, 20 μM MG-132, 2 μM matrix metalloprotease inhibitor III (444264, Calbiochem, San Diego, USA), and 1 % protease inhibitor cocktail III (539134, Calbiochem, San Diego, USA). Homogenization was carried out at 4°C with a Tekmar homogenizer by three 15 s bursts with a minute cooling in-between. Homogenates were centrifuged at 4°C for 1 h at 100 000 × g. The supernatant solutions are referred to as "soluble" fractions. The pellets were suspended in the 0.2-0.25 ml complete buffer containing 1 % ASB-14 and were mixed every 15 min for 2 h with Radnoti glass pestles (Unitek, Monrovia, USA). After centrifugation at 4°C for 1 h at 100 000 × g, the supernatants, referred to as "membrane extracts", were removed and the pellets were discarded. About 50 oocytes of each soluble and membrane extract for each gene testing were separated by lithium dodecyl sulfate polyacrylamide gel electrophoresis on 4-12 % Bis-Tris NuPAGE gels and transferred to PVDF membranes (Invitrogen, Carlsbad, USA); nonspecific binding was blocked by overnight incubation in 1 % casein in PBS at room temperature. Blots were then probed for 2-4 h at room temperature with antibodys of PDIA3 (anti-PDIA3; ab13507, abcam, Cambridge, USA), SOD1 (anti-SOD1; ab13498, abcam, Cambridge, USA) and VIM (anti-VIM; V6630, Sigma, USA). Histone H2B (anti-H2B; ab40975, abcam, Cambridge, USA) served as loading control. After 2 h incubation at room temperature with secondary antibodies, protein bands were detected by enhanced chemiluminescence with the RPN2108 kit (Amersham, Piscataway, USA) and Kodak BioMax Light film.

Statistical analysis
Statistical analysis was performed using SPSS 13.0 for MicroSoft TM Windows. Data are shown as the mean ± SD. One-way ANOVA was used to assess any differences between groups. The Duncan method was employed for pairwise comparisons, followed by a Bonferroni correction. p < 0.05 (two-tailed) was considered statistically significant.

Maturation of porcine oocytes during IVM
To get the time lapse characteristics of maturation of porcine oocytes during IVM, we detected the rate of 1 st polarbody extrusion from 16 h to 42 h. The oocyte began to extrude polarbody mainly at 25 h (14.45 %) of IVM and reached a plateau at 33 h (Fig. 1), and there was no significant difference between the rates at 33 h (76.72 %) and 42 h (81.80 %) of IVM (Additional file 1: Table S2; p > 0.05). We also found most of 33O (79.71 %) were arrested at the MII stage, and it had no remarkable difference with 42O (83.86 %; Table 1). Theoretically, the maturation quality of 42O must be better than 33O [17][18][19][20][21].

Effects of 33O and 42O on porcine embryo development
In order to test the effect of maturation time on nuclear reprogramming and embryo development, we employed 33O and 42O to produce IVF, SCNT and PA embryos ( Table 2). The results showed the rates of cleavage and blastocyst of IVF embryos from 33O were significant higher than that from 42O (78.36 % vs 67.22 %, p < 0.05; 33.79 % vs 19.73 %, p < 0.05, respectively). Also, the rate of fertilization of 33O was significantly enhanced compared to 42O (85.19 % vs 78.09 %, p < 0.05; Additional file 1: Table S3). On the contrary, the rates of cleavage and blastocyst of SCNT embryos from 42O were significant higher than that from 33O (89.17 % vs 67.60 %, p < 0.05; 22.34 % vs13.54 %, p < 0.05, respectively), and the rates of enucleation of oocyte in both 33O and 42O reached up to 90 % (Additional file 1: Table S4), excluding the influence of enucleation on cloned embryo development. Moreover, the rates of cleavage and blastocyst of PA embryos from 42O were also significant higher than that from 33O (92.23 % vs 43.74 %, p < 0.05; 43.64 % vs 17.84 %, p < 0.05), and there was no significant difference of pronuclear rates at 6 h after artificial activation between 33O and 42O (Additional file 1: Table S5), suggesting lower development of 33O may be not simply due to lower activation compared to 42O. These results indicate that further maturation of oocyte after 1 st polarbody extrusion can accumulate maternal factors for SCNT and PA embryos development, but it may be not necessary for IVF embryo.

Functional categorization and pathway analysis of the identified factors
We categorized proteins identified in this study by searching Gene Ontology and performing a literature search. The differentially expressed proteins were classified into groups based on molecular function (structural constituent of cytoskeleton, unfolded protein binding, nucleotide binding, NAD or NADH binding, ribonucleotide binding, calcium ion binding and ATP binding), biological process (cellular homeostasis, response to inorganic substance, response to drug, intermediate, cell death, cellular ion homeostasis, maintenance of location, antioxidation and monosaccharide catabolic), and cellular component (cytoplasm, membrane, endoplasmic reticulum, cytoskeleton, cytosol, perinuclear region of cytoplasm and nucleus) with the UniProt database (Fig. 5). DAVID and Agilent Literature Search were combined by cytoscape to cluster most affected Values with different superscripts in the same group differ significantly (P < 0.05) Fig. 2 Total proteins seperated by 2D DIGE. Samples prepared from 33O (a1, b2) and 42O (a2, b1) were labeled with Cy3 or Cy5, separately, and the mixed samples were labeled with Cy2 for standard protein (a3, b3). The blue spots are internal standard proteins consisting of all samples labeled with Cy2. The differentially expressed protein spots between 33O and 42O were numbered in the 2D DIGE image (c) molecular functions in oocyte maturation, namely embryonic development, embryogenesis, ovulation, epigenetic modification, fertilization, meiosis, ageing, chromatin remodeling and anti-ageing. Additionally, we employed a detailed analysis of embryonic development, using automated text-mining tool which enables the software to generate pathways from entries in the PubMed database and Gene Ontology. These analysis revealed that proteins related to fertilization were found to be highly synthesized in 33O, such as PDIA3 [16], and nuclear reprogramming was significantly affected by high expressed proteins in 42O, such as PARK7 that was proved to be required for successful development of porcine SCNT embryos [22].

Discussion
An increasing amount of data demonstrate that oocyte maturation involves major changes within the cytoplasmic compartment and these changes are essential for nuclear reprogramming and embryo development [14]. However, the molecular factors remain largely unknown.
In the study, we found that 33O and 42O exhibited distinct developmental competences after IVF, SCNT and PA. Additionally, 18 differentially expressed proteins between 33O and 42O were identified by MALDI-TOF/ MS in porcine oocytes. Insufficient maturation of oocyte is not enough to support the subsequent embryo development, and oocyte will be senescent if overmatured [23]. It has been generally considered that porcine oocyte reaches full maturation at 42 h of IVM, and a number of research groups including us use oocytes matured at 42 h for IVF, PA and SCNT study [17][18][19][20][21]. In the study, we demonstrated the rate of 1 st polarbody extrusion of porcine oocytes reached a plateau at 33 h of IVM and the most part of the oocytes arrested at the MII stage. The result was not consistent with a previous report that described porcine oocyte arrived at MII stage during 34.4-48 h of IVM [24], and this may result from the addition of EGF to the IVM medium in the experiments for this research, which may have accelerated the nuclear maturation as already observed in other study [25].
Oocyte maturation is among the most crucial factors affecting embryo development [26][27][28][29]. So, we described the effects of 33O and 42O on embryo development of IVF, SCNT and PA. In consistent with previous reports [26][27][28], the developmental competence of SCNT and PA embryos by 42O were markedly improved in comparison with 33O, indicating that full cytoplasmic maturation is indispensable for development of SCNT and PA embryos. However, surprisingly, 33O was able to sustain IVF embryo development with higher competence comparing to 42O. This is in line with previous results showing that a suitable culture period for IVM of porcine oocytes to IVF is 32 to 36 h [30]. These results could be explained by the following reasons: firstly, according to our results, the rate of fertilization of 33O was higher, so more zygotes may initiate subsequent embryo development; secondly, the nuclear reprogramming of sperm and somatic cell undergoes two distinct processes. The success of SCNT depends on several parameters that impact on the ability of oocyte to remodel the nuclear morphology of donor cell, to reverse the epigenetic modifications to totipotent status, and to activate the embryonic gene expression required for further Fig. 3 Differentially expressed proteins identified between 33O and 42O. a All protein spots (994) from 2D DIGE analysis. A horizontal axis represents the average log2 (ratio) of proteins (42O/33O), whereas a vertical axis represents the -log10 (P-value), P-value from t-test, red plots represent the protein spots which were significantly changed (fold > 1.5 and p < 0.05). b Heat map generated from 20 protein features selected in the 2D DIGE for analysis development [31]. So, the great requirement of reprogramming factors for SCNT must be different with IVF and 42O is rich in these factors [32,33]; thirdly, the stresscoping mechanisms are probably relying on different molecules between SCNT and IVF embryos. This notion is supported by the fact that SCNT embryos exhibit higher rates of apoptosis, weaker stress-coping functions and are less tolerant to the in vitro culture environment than IVF embryos [34]. Lower level of gene expression related to cell protection from stress and apoptosis has been reported in oocyte with low cytoplasmic maturation quality [35], such as SOD1, one of the major antioxidant enzymes, which was proved to scavenge superoxide during early embryonic development [36,37], which was found abundantly in 42O by our proteome analysis. Considering that, it is reasonable to deduce that transferred somatic nuclei undergo lower cellular stress than sperms in 42O, so that 42O can support better development of SCNT embryos.
During the last decade, several approaches analyzing the protein composition of oocyte have been conducted including the proteome signature of porcine oocyte [6][7][8][9][10][11][12][13][14][15], however, the disadvantage of these proteome analyses is that no factor related to nuclear reprogramming and embryo development has been described. In the study, efforts have been made to discover differentially expressed proteins between 33O and 42O by proteome analysis. 994 unique protein spots were detected by 2D DIGE, and only 25 protein spots were found to be differentially expressed between 33O and 42O. That is to say, the difference was no more than 2 % (25/994), indicating the proteome signature of 33O and 42O were very similar. But the developmental competence of 33O and 42O were largely different, suggesting that the few differential proteins identified played important roles in embryo development. This is in line with previous results that only a few proteins which are indispensable for embryo development are largely synthesized from stored mRNAs in the final stage of oocyte maturation [32,33].
To further clear the molecular basis for cytoplasmic maturation, we focused on function analysis of these identified proteins. Proteins, such as HSP90B1, HSPA5 and HSP90AB1, with chaperone function were most commonly found, reflecting a marked change of nuclear proteins during oocyte maturation [7,13]. Cluster analysis of the 18 proteins with the UniProt database showed a clear trend towards molecular functions and biological processes related to protein synthesis, including ribonucleotide binding, nucleotide binding and cellular homeostasis, that comfirm the points that accumulation of proteins happen constantly during cytoplasmic maturation [7,13]. Moreover, the influence of the differential expression factors on development of IVF and SCNT embryos was unraveled by pathway analysis of these proteins using DAVID and Agilent Literature Search. INPP1 and PDIA3 are predominantly involved in fertilization and were found to be over-expressed in 33O. Also, a characterized nuclear reprogramming factor, PARK7, was more abundant in 42O [22]. This led us to suggest that the 18 identified proteins were mainly responsible for nuclear reprogramming and early embryo development.

Conclusions
In summary, the study identifyed the differentially expressed proteins between porcine oocytes during 33 h and 42 h of IVM. We showed that majority of porcine oocytes have achieved nuclear maturation at 33 h of IVM, and there are 18 protein factors related to nuclear reprogramming and embryo development discovered by proteome analysis of 33O and 44O. Analysis of these factors indicates several of them may be responsible for the development competence difference between 33O and 42O when used for IVF and SCNT. Our study