Introduction
In vitro-produced embryos have a reduced competence with a negative effect on the production of a viable offspring. The lower quality of these embryos is due to changes in gene expression patterns as a result of interaction with the adverse environment generated by in vitro conditions (in vitro oocyte maturation, in vitro fertilization, embryo culture system, etc.) (Assou et al., Reference Assou, Boumela, Haouzi, Anahory, Dechaud, De Vos and Hamamah2011; Cánepa et al., Reference Cánepa, Ortega, Monteleone, Mucci, Kaiser, Brocco and Mutto2014). Morphological examination in bovine is currently the ‘gold standard’ for assessment of embryo quality, nevertheless this is a subjective judgement that does not necessarily reflect intrinsic embryonic competence (Van Soom et al., Reference Van Soom, Mateusen, Leroy and De Kruif2003; Hasler, Reference Hasler2014). The combination of morphological scoring and screening of gene markers might be a powerful tool to select the most competent embryos. However, selection of gene markers has been a very complicated task as many signalling pathway are activated during embryo development and many genes might be aberrantly expressed in in vitro-produced embryos (Niwa et al., Reference Niwa, Miyazaki and Smith2000; Boiani et al., Reference Boiani, Eckardt, Schöler and McLaughlin2002; Wrenzycki et al., Reference Wrenzycki, Herrmann, Lucas-Hahn, Korsawe, Lemme and Niemann2005; Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). This fact makes it difficult to define which genes are the master regulators of normal early embryo development and, furthermore, which genes are responsible for controlling or ensuring long-term development.
Genes related to first cell lineage segregation, and contributing to form the inner cell mass (ICM) and trophoblast (TB) cells in the blastocyst, are crucial for embryo development after this stage (Ozawa et al., Reference Ozawa, Sakatani, Yao, Shanker, Yu, Yamashita, Wakabayashi, Nakai, Dobbs, Sudano, Farmerie and Hansen2012). Pluripotency markers, OCT4, SOX2 and NANOG have been defined in mouse, and are crucial for normal control of pluripotency pathways in preimplantation embryos (Marikawa and Alarcón, Reference Marikawa and Alarcón2009). Mouse embryos lacking OCT4 may have normal blastocysts morphology but the ICM differentiates into the trophectoderm and therefore is not developmentally competent (Nichols et al., Reference Nichols, Zevnik, Anastassiadis, Niwa, Klewe-Nebenius, Chambers, Schöler and Smith1998; Niwa et al., Reference Niwa, Miyazaki and Smith2000; Frum et al., Reference Frum, Halbisen, Wang, Amiri, Robson and Ralston2013). Similarly, mouse embryos with a homozygous deletion of SOX2 develop to the blastocyst stage but fail to implant (Avilion et al., Reference Avilion, Nicolis, Pevny, Perez, Vivian and Lovell-Badge2003). NANOG is restricted to a subset of cells in the ICM and localizes in the epiblast before implantation of mouse embryos. It seems to be the signal to maintain FGF4 expression by the epiblast (Chazaud et al., Reference Chazaud, Yamanaka, Pawson and Rossant2006).
These three pluripotency markers have been found aberrantly expressed in in vitro embryos produced by IVF or SCNT (Dean et al., Reference Dean, Santos, Stojkovic, Zakhartchenko, Walter, Wolf and Reik2001; Wrenzycki et al., Reference Wrenzycki, Wells, Herrmann, Miller, Oliver, Tervit and Niemann2001; Beyhan et al., Reference Beyhan, Ross, Iager, Kocabas, Cunniff, Rosa and Cibelli2007; Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Cox, Tovar, Einspanier and Castro2010, Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). Aberrant OCT4 and SOX2 expression in blastocysts has been related to a low ability of cloned embryos to elongate in vivo and produce live offspring (Hall et al., Reference Hall, Ruddock and French2005; Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Cox, Tovar, Einspanier and Castro2010, Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). However, when cloned blastocysts produced under the same conditions were transferred to surrogates and recovered at elongation, no differences in OCT4 and SOX2 expression levels between clones and IVF-derived controls were observed (Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Cox, Tovar, Einspanier and Castro2010). This result led to the hypothesis that only blastocysts with certain levels of OCT4 and SOX2 expression are most likely to reach the elongation stage (Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Cox, Tovar, Einspanier and Castro2010, Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). Furthermore, bovine blastocysts with a higher expression of OCT4 had a better morphology with higher cell numbers (Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). Recently, it was shown that OCT4 knockout bovine embryos do not express NANOG and that their development is blocked at the early blastocyst stage (Simmet et al., Reference Simmet, Zakhartchenko, Philippou-Massier, Blum, Klymiuk and Wolf2018; Daigneault et al., Reference Daigneault, Rajput, Smith and Ross2018).
The data presented indicated that expression of canonical pluripotency markers is critical for embryo competence and further development. Most studies have been carried out in mice and very little information is known about the effect of this gene expression on the developmental competence of embryos from other species. Moreover, in all reports, the entire embryo was used for gene expression analysis at the blastocyst stage, even though all embryos are very different in terms of competence (Bertolini et al., Reference Bertolini, Beam, Shim, Bertolini, Moyer, Famula and Anderson2002; Ozawa et al., Reference Ozawa, Sakatani, Yao, Shanker, Yu, Yamashita, Wakabayashi, Nakai, Dobbs, Sudano, Farmerie and Hansen2012); therefore it is likely that the group of studied blastocysts did not reflect embryos selected for transfer. In this work, we proposed an experiment to test the effect of OCT4, SOX2 and NANOG expression levels on the developmental competence of in vitro-derived bovine embryos. A model was designed using blastocyst splitting to derived two identical demi-embryos, so that one half could be used for gene expression analysis, while the other half was kept in culture to determine developmental competence and any possible correlation with the expression levels of pluripotency markers.
Materials and Methods
All reagents and media except otherwise stated were from Sigma Chemical Co. (St. Louis, MO, USA).
Experimental design
The main goal of this work was to evaluate the effect of the expression level of pluripotency markers OCT4, SOX2 and NANOG at the blastocyst stage on the developmental potential of the bovine embryo during the peri-implantation period. Because there is a high variability between embryos even produced under the same conditions, a model was proposed using blastocyst splitting to derive two similar demi-embryos in which one could be a ‘mirror’ image of the other to investigate further developmental competence of the counterpart. To test peri-implantation development, an extended culture system up to D13 of development was used to follow embryo development and morphological changes. An extended in vitro system was used for practical reasons to increase the number of evaluated embryos and avoid costs associated with individual embryo transfer and variability due to the maternal environment. Grade I hatched D8 blastocyst were selected for this experiment based on previous results (Velásquez et al., Reference Velásquez, Castro, Veraguas, Cox, Lara, Briones and Rodríguez-Alvarez2016). After splitting, embryos were kept in culture for 6 h to confirm their viability; only those couples of demi-embryos with a similar morphology (visible ICM and a diameter difference <20%) were selected. From each couple, one D9 embryo was kept frozen for gene expression analysis, while the counterpart was kept in culture until D13 of development. Day 9 embryos (frozen counterpart) were assigned a posteriori to one of the following groups: G1, demi-embryos whose counterpart did not develop until D13 of in vitro culture; or G2, demi-embryos whose counterpart developed until D13, showing an expanded morphology without degeneration signs and diameter similar to that of non-split embryos, previously reported by our group (Velásquez et al., Reference Velásquez, Manríquez, Castro, Cox and Rodríguez-Alvarez2017).
The expression levels of OCT4, SOX2 and NANOG were determined in D9 demi-embryos from both groups (G1 and G2) and the expression levels were correlated with the developmental potential of the counterpart. A second analysis was performed to determine the significance of the expression levels of the pluripotency markers at D9 (from G2) and the expression of crucial genes in their D13 counterpart. A chart of experimental design is presented in Fig. 1.
Representative chart of experimental design. D9 demi-embryos were assigned to two groups depending on the in vitro development of their counterpart; G1 are those D9 demi-embryos whose counterpart developed up to D13, while G2 are those D9 demi-embryos in which the counterpart did not develop up to D13.
In vitro blastocyst production
Bovine embryos were produced by in vitro fertilization (IVF). Cattle oocytes were obtained from ovaries collected in a local slaughterhouse and in vitro matured (IVM) following standard protocols described elsewhere (Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). Cumulus–oocyte complexes (COCs) were in vitro matured in TCM-199 Earle’s salts, supplemented with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (0.01 U/ml each), 17β-estradiol (1 μg/ml), epidermal growth factor (EGF; 10 ng/ml), and 10% fetal bovine serum (FBS) and placed at 39°C in an incubator in a 5% CO2 in air atmosphere. After 21 h of IVM, matured oocytes were in vitro fertilized with thawed semen from a single bull of proven fertility. The motile fraction of the semen was obtained using a Percoll gradient (45% Percoll over 90% Percoll) and centrifuged at 1200 rpm for 25 min (Velásquez et al., Reference Velásquez, Castro, Veraguas, Cox, Lara, Briones and Rodríguez-Alvarez2016). Approximately 1 × 106 motile sperms/ml were used to fertilize 25 COCs in 500 µl of TALP medium supplemented with 2 mM sodium pyruvate, 50 μg/ml gentamycin, 0.01 mg/ml heparin sodium salt, and 6 mg/ml of bovine serum albumin (BSA) fraction V (essentially fatty acid free). Fertilization was carried out for 24 h at 39°C in a 5% CO2 in air atmosphere. After IVF, presumptive zygotes were denuded by 3 min vortexing in TCM-Hepes medium + 0.3 mg/ml of hyaluronidase. Embryos were cultured in groups of 25–30 per well in synthetic oviduct fluid (SOFaa) supplemented with 0.37 mM trisodium citrate, 2.77 mM myo-inositol, essential and non-essential amino acids (final concentration 1×), gentamycin (50 μg/ml), 3 mg/ml essentially fatty acid free BSA, 2% FBS and 10 ng/ml EGF. Culture dishes were placed in sealed aluminium foil bags containing a balanced gas atmosphere consisting of 5% CO2, 5.5% O2 and 89.5% N2, 100% humidity at 39°C. At day 7 of culture, bags were opened, then blastocysts were counted and classified as grade I, II, or III in agreement with ICM and trophoblast quality. Day 0 of embryo development corresponded to the day of IVF.
Embryo splitting
Grade I bovine hatched D8 blastocysts, those with a visible blastocoel cavity, forming many closely aggregated cells a well developed ICM and a trophoblast formed by a very well defined layer of lengthened cells, were used for splitting. In a previous study, it was demonstrated that splitting D8 embryos generated identical blastocysts with similar gene expression patterns (Velásquez et al., Reference Velásquez, Castro, Veraguas, Cox, Lara, Briones and Rodríguez-Alvarez2016). The split was performed manually with a micro-blade under a stereoscopic microscope (Ultra Sharp Splitting Blades; AB Technology, Pullman, WA, USA) following the protocol established previously in our laboratory (Velásquez et al., Reference Velásquez, Castro, Veraguas, Cox, Lara, Briones and Rodríguez-Alvarez2016). Splitting was performed in D8 hatched blastocysts to obtain the highest rate of demi-embryo survival (Velásquez et al., Reference Velásquez, Castro, Veraguas, Cox, Lara, Briones and Rodríguez-Alvarez2016). Hatched blastocysts were randomly selected for splitting or for the control group.
After splitting, demi-embryos were kept in culture in SOFaa for an additional 6 h to confirm embryo survival and quality. Splitting was considered successful when both demi-embryos survived and showed similar morphology as described earlier. One demi-embryo from each couple was selected for in vitro embryo culture up to D13, while the counterpart was selected for gene expression analysis.
Extended in vitro culture of blastocyst up to D13 of in vitro development
Individual demi-embryos were in vitro cultured up to D13 of embryo development in a four-well dish (Nunc, Roskilde, Denmark) in SOFaa, following the protocols standardized by our group (Velásquez et al., Reference Velásquez, Manríquez, Castro, Cox and Rodríguez-Alvarez2017). To verify embryo viability, the diameter and morphological characteristics of each embryo were measured daily, using 5-Mpx Micrometrics digital camera software (Arquimed, Santiago de Chile, Chile). Embryo viability was determined by the lack of obvious cell degeneration and lineal growth during the extended culture. At D13, attached or obviously dead embryos were excluded from further analyses.
RNA extraction and qRT-PCR analysis
D9 and D13 individual embryos were treated with the Cells-to-cDNA TM II kit (Ambion Co., Austin, TX, USA) lysis buffer according to the manufacturer’s instruction. Briefly, embryos were washed in cold phosphate-buffered saline (PBS) and then 100 µl of lysis buffer were added and embryos were incubated for 10 min at 75°C. All samples were treated with DNase I (0.04 U/µl) for genomic DNA digestion. For cDNA conversion, 10 µl of total RNA was used in a 20-µl final reaction containing 5 µM random primers, 10 mM each of dNTPs, 2 µl first strand buffer (10×), 10 U of RNase inhibitor, and 200 U/ml M-MuLV (Ambion Co., Austin, TX, USA). Cycling parameters were: 70°C for 3 min, 42°C for 60 min, and 92°C for 10 min. The produced cDNAs were kept frozen at −20°C until PCR analysis.
Gene expression analysis was performed using real-time PCR (RT-PCR) and the standard curve method. Standard curves for each gene were prepared using PCR products excised and eluted from agarose gels using a gel extraction kit (Omega Biotek, Santiago, Chile) and quantified by Epoch. Serial 10-fold dilutions of PCR products were prepared, starting for 1 ng of amplified DNA. Eight points were included in each standard curve, and 2 µl aliquots were used in duplicate for each point of the curve. For RT-PCR, samples were loaded in duplicate (technical replicates). The primers used and PCR conditions for each gene are listed in Table 1. The crossing point (CP) and the amplification efficiency were calculated using integrated software.
Oligonucleotide composition and PCR conditions for gene expression analysis
At D9, only pluripotency markers OCT4, SOX2 and NANOG were evaluated, while at D13 TP1, EOMES, FGF4, CDX2 and TKDP1 were also analyzed. In all cases, ACTB was used as the internal control, based on our previous experience using this gene as the housekeeping gene (Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013; Velásquez et al., Reference Velásquez, Manríquez, Castro, Cox and Rodríguez-Alvarez2017).
Statistical analysis
Correlation analyses were performed using Pearson’s correlation test while gene expression level was analyzed between groups by the non-parametric Wilcoxon test. A P-value < 0.05 was considered to be statistically significant. The software used was InfoStat (Buenos Aires, Argentina).
Results
To test the hypothesis, 59 blastocysts at D8 of culture were split to generate 26 couples of demi-embryos with similar morphology regarding diameter and presence of the ICM (diameter differences less than 20%). From each couple of demi-embryos, one of these was randomly selected for gene expression analysis (D9 blastocysts), while its counterpart was kept in an extended individual culture system until D13. From the 26 cultured demi-embryos, 11 continued their development until D13 without sign of degeneration and with linear growth, reaching D13, with a diameter similar to non-split embryos evaluated in a previous study (split: 368 ± 119.1 µm; non-split 532.2 ± 214.8 µm (Velásquez et al., Reference Velásquez, Manríquez, Castro, Cox and Rodríguez-Alvarez2017); P > 0.05). The other 15 embryos degenerated before D13, observed as a compact, dark mass of cells. The demi-embryos at D9 used for gene expression analysis were divided a posteriori in two groups as mentioned before: G1, degenerated counterpart; and G2, developed counterpart, so D9 embryos were defined as competent embryos.
In the first analysis, the expression levels of OCT4, SOX2 and NANOG were compared between D9 blastocysts from G1 and G2. The expression of SOX2 and NANOG was significantly higher in demi-embryos whose counterpart did not developed properly in vitro (G1) (P < 0.05) (Fig. 2). This finding is in agreement with the negative correlation observed between the expression of SOX2 in D9 demi-embryos and the survival of their counterpart up to D13 (r = −0.37, P = 0.03). Furthermore, we evaluated the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2. This evaluation showed that embryos from G2, considered as competent, had a quotient close to one (SOX2/OCT4: 1.1), while the other group (G2) had a quotient of 2.3 due to a higher expression of SOX2 in these embryos.
Relative gene expression analysis of the pluripotency markers in D9 embryos; G1, those whose counterpart did not develop; and G2, those embryos whose counterpart developed up to D13 (competent embryos). *Indicates Pearson’s correlation coefficient and significance from the analysis between the SOX2 expression level and embryo survival up to D13 of in vitro development. Different letters within a same graph indicate significant differences (P < 0.05).
In a second analysis, D9 blastocyst from G2 were excluded and the expression levels of pluripotency genes were compared between D9 and D13 embryos. The expression of the three markers decreased significantly from D9 to D13 (Fig. 3). In addition, two correlation analyses were performed, the first between the relative expression of the pluripotency markers D9 blastocyst and embryo diameter at D13. While the second was realized between the expression levels of pluripotency markers for D9 blastocysts and the expression of the trophoblastic markers in their counterpart at D13 (Table 2). The first analysis indicated a positive correlation between the expression of OCT4 and the diameter of D13 embryos. Furthermore, the second analysis indicated a positive correlation between the expression of SOX2 at the blastocyst stage and the expression of TP1 at D13, and positive correlation between the expression of NANOG in the blastocyst and the expression of EOMES and FGF4 at D13 (Table 2).
Relative gene expression analysis of the pluripotency markers in D9 demi-embryos and their counterpart with development up to D13. P-values indicate statistical significance in all cases.
Gene expression correlation analysis of the pluripotency markers at D9 of in vitro development with the embryo diameter and the trophoblastic markers expression in their counterparts at D13 of in vitro development
NC: No correlation.
Discussion
Morphology alone is not an adequate criteria to evaluate embryo competence (Wrenzycki et al., Reference Wrenzycki, Herrmann, Lucas-Hahn, Lemme, Korsawe and Niemann2004; Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). During early development, the expression of specific genes at every phase promotes embryo progression from one stage to the next (Hamatani et al., Reference Hamatani, Carter, Sharov and Ko2004). For this reason, the relative quantification of mRNA of specific genes during early development is a more appropriate indicator of embryo quality (Wrenzycki et al., Reference Wrenzycki, Herrmann, Lucas-Hahn, Lemme, Korsawe and Niemann2004; Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Cox, Tovar, Einspanier and Castro2010, Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013; Velásquez et al., Reference Velásquez, Castro, Veraguas, Cox, Lara, Briones and Rodríguez-Alvarez2016).
In the present research, the effect of expression level of pluripotency markers OCT4, SOX2 and NANOG, at blastocyst stage, on embryo development up D13 of embryo development was evaluated. A model using twins embryos produced by splitting bovine blastocysts to analyse gene expression at D9 and D13 was proposed. This model allowed the production of two similar demi-embryos, one for gene expression analysis at D9, while the other demi-embryo could continue its development, reducing the variability caused by using individual embryos.
Reports from mouse embryos have pointed out that OCT4 is the main regulator of cell pluripotency (Wu and Schöler, Reference Wu and Schöler2014). Different reports have described that the development of mouse embryos beyond the blastocyst stage depends on OCT4 expression (Nichols et al., Reference Nichols, Zevnik, Anastassiadis, Niwa, Klewe-Nebenius, Chambers, Schöler and Smith1998). In mouse, OCT4 is exclusively expressed in the ICM (Boiani et al., Reference Boiani, Eckardt, Schöler and McLaughlin2002) however, in bovine, OCT4 expression is not restricted to the ICM and its protein levels remain high in TB cells during the blastocyst stage (Kirchhof et al., Reference Kirchhof, Carnwath, Lemme, Anastassiadis, Schöler and Niemann2000). Recent studies have demonstrated that, in bovine embryos, OCT4 is not required for embryo transition from morula to blastocyst stages (Daigneault et al., Reference Daigneault, Rajput, Smith and Ross2018; Simmet et al., Reference Simmet, Zakhartchenko, Philippou-Massier, Blum, Klymiuk and Wolf2018). It seems that lack of OCT4 affects normal expression of CDX2 but it is still expressed in the TB cells of bovine blastocysts (Daigneault et al., Reference Daigneault, Rajput, Smith and Ross2018; Simmet et al., Reference Simmet, Zakhartchenko, Philippou-Massier, Blum, Klymiuk and Wolf2018). SOX2 expression is not affected by lack of OCT4 (Daigneault et al., Reference Daigneault, Rajput, Smith and Ross2018), but OCT4 is required for NANOG expression (Simmet et al., Reference Simmet, Zakhartchenko, Philippou-Massier, Blum, Klymiuk and Wolf2018). Both markers SOX2 and NANOG are highly expressed in cells of the ICM during the blastocyst stage in mice (Avilion, et al., Reference Avilion, Nicolis, Pevny, Perez, Vivian and Lovell-Badge2003), humans (Adjaye, Reference Adjaye2005) and bovine embryos (Ozawa et al., Reference Ozawa, Sakatani, Yao, Shanker, Yu, Yamashita, Wakabayashi, Nakai, Dobbs, Sudano, Farmerie and Hansen2012; Zhao et al., Reference Zhao, Cui, Hao, Wang, Zhao, Du, Wang, Liu and Zhu2016). The absence of SOX2 promotes the differentiation of stem cells to cells that are similar to trophoblasts (Masui et al., Reference Masui, Nakatake, Toyooka, Shimosato, Yagi, Takahashi, Okochi, Okuda, Matoba, Sharov, Ko and Niwa2007).
The mentioned reports indicated that OCT4, SOX2 and NANOG are crucial for the normal development of bovine embryos beyond the early blastocyst stage and even the downregulation of these genes might reduce embryo developmental competence (Beyhan et al., Reference Beyhan, Ross, Iager, Kocabas, Cunniff, Rosa and Cibelli2007; Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013). However, very little information is known regarding the effect of upregulation on the expression of pluripotency markers in bovine blastocysts. In this study, OCT4 expression was similar between competent (those whose counterpart developed up to D13) and non-competent (did not develop further) D9 blastocysts, but the expression levels of NANOG and SOX2 were higher in non-competent embryos. Furthermore, competent embryos had similar OCT4 and SOX2 expression levels with a SOX2/OCT4 ratio of 1.1, whereas this ratio was double in non-competent D9 embryos. This finding might indicate that a balance between OCT4 and SOX2 expression is required for bovine embryo development, most likely to maintain normal regulation of pluripotency.
Yamaguchi et al. (Reference Yamaguchi, Hirano, Nagata and Tada2011), evaluated the SOX2 effect on reprograming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). It was observed that transfection with SOX2 at a high dose resulted in a decreased expression of OCT4 in these cells (Yamaguchi et al. Reference Yamaguchi, Hirano, Nagata and Tada2011). This result suggest that overexpression of SOX2 affected the reprograming capacity, therefore a correct SOX2 expression might be essential to induce efficient reprograming to generate iPSCs. Pan and Schultz (Reference Pan and Schultz2011) reported similar results; in their research, complementary RNA of SOX2 was microinjected into 1-cell mouse embryos to generate overexpression of this gene. Here, 70% of the injected embryos overexpressed SOX2 and suffered developmental arrest at the 2-cell stage due to repression of 15% of the genes in zygotes (Pan and Schultz, Reference Pan and Schultz2011).
Furthermore, Moro et al. (Reference Moro, Hiriart, Buemo, Jarazo, Sestelo, Veraguas, Rodríguez-Alvarez and Salamone2015) reported that cat blastocysts generated by SCNT showed overexpression of the genes OCT4, SOX2, NANOG and CDX2 compared with cat blastocysts generated by IVF. These results indicated that the technique used for in vitro embryo production affected significantly the gene expression pattern of the embryos. Furthermore, it was reported that bovine embryos with a decreased developmental capability had increased transcription rates of the pluripotency markers, with unbalanced expression of these genes (Khan et al., Reference Khan, Dubé, Gall, Peynot, Ruffini, Laffont, Le Bourhis, Degrelle, Jouneau and Duranthon2012). This aberrant gene expression was not reflected in embryo morphology, as these embryos had apparently normal development in vitro. However, this overexpression might have negative effects on the later stages of development, and could generate anomalies or developmental arrest in the long term (Khan et al., Reference Khan, Dubé, Gall, Peynot, Ruffini, Laffont, Le Bourhis, Degrelle, Jouneau and Duranthon2012).
In addition, we performed a correlation analysis between the expression pattern of the pluripotency markers in D9 competent embryos and embryo quality at D13 (embryo diameter and gene expression pattern). We saw a positive correlation (r = 0.6; P = 0.01) between OCT4 expression and embryo diameter. This finding is in agreement with previous research carried out in equine embryos that described that the expression levels of all studied pluripotency markers (OCT4, NANOG, DPPA4, GDF3 and TERT) were increased in embryos with a larger diameter (Paris et al., Reference Paris, Kuijk, Roelen and Stout2008). Furthermore, in a study in which bovine embryos were generated by SCNT, positive correlation between OCT4 expression in the blastocysts and their total cell number (r = 0.98; P = 0.002) was observed that was closely related to the embryo diameter (Rodríguez-Alvarez et al., Reference Rodríguez-Alvarez, Manríquez, Velásquez and Castro2013).
There was also a correlation between the expression of pluripotency markers in D9 competent embryos and the expression of TB markers. SOX2 expression correlated with the expression of interferon-tau (TP1) at D13 of development. This finding might indicate that an adequate expression of SOX2 generates an increase in TP1 expression in the trophoblast that is essential for correct embryo–maternal recognition, as TP1 is the main marker of pregnancy recognition in bovine (Viebahn, Reference Viebahn1999; Degrelle et al., Reference Degrelle, Campion, Cabau, Piumi, Reinaud, Richard, Renard and Hue2005; Roberts et al., Reference Roberts, Chen, Ezashi and Walker2008). NANOG expression at D9 correlated with the expression of EOMES and FGF4 at D13 of development. These pluripotency markers are generally considered inhibitors of the differentiation process. However, a study performed by Teo et al. (Reference Teo, Arnold, Trotter, Brown, Ang, Chng, Robertson, Dunn and Vallier2011) showed that NANOG participates actively in differentiation of the endoderm, promoting EOMES expression and is in agreement with our results. These results indicated that the expression level of pluripotency markers at D9 determined further embryo development, and the quality and functionality of trophoblast to mediate embryo–maternal cross-talk.
Finally, we observed a significant decrease in the expression levels of OCT4, SOX2 and NANOG at D13 of development. Previously, Degrelle et al. (Reference Degrelle, Campion, Cabau, Piumi, Reinaud, Richard, Renard and Hue2005), described that OCT4 expression was silenced at the end of the elongation stage in bovine embryos, while Rodríguez-Alvarez et al. (Reference Rodríguez-Alvarez, Cox, Tovar, Einspanier and Castro2010) indicated that OCT4 expression remained constant between D7 and D17 of bovine embryo development. However, in those reports, embryos analyzed at the blastocyst stage were not the same embryos analyzed at the elongation stage. In the present research, the D13 embryo might be considered as the same embryo at D9.
The results obtained indicated the presence of a threshold in the expression of the pluripotency markers that, when exceeded, generated a negative effect on embryo development. The coordinated expression of SOX2 and OCT4 is crucial for bovine embryo development. In this study, the embryos that accomplished adequate development had a strictly equilibrated gene expression pattern. It seems that embryo manipulation induced overexpression of the pluripotency markers, probably as a compensatory mechanism that later resulted in abnormal peri-implantation embryo development. There has been recent information regarding the role of OCT4, indicating that this marker is not required for SOX2 expression during early development of bovine embryos, but is still unknown if SOX2 is the master regulator of pluripotency in bovine embryos.
Financial support
This work was supported by grant: FONDECYT no. 1170310 and CONICYT national doctoral scholarship no. 21140234 (Ministry of Education) Chile.
Statement of interest
The authors declare no competing interests.
Ethical standards
The study was approved by the Ethics Committee at the Faculty of Veterinary Sciences, Universidad de Concepcion, Chile (permit no. CBE-17-2017).
