Introduction
It is well known that the first embryonic cell lineage differentiation, or, the emergence of trophectoderm (TE) and inner cell mass (ICM), is a key event in preimplantation development of mammalian embryo. The differentiation of cells into ICM and TE is linked closely to the specification of the blastocyst embryonic–abembryonic (Em–Ab) axis. Until now, there are two conflicting views on the establishment of the Em–Ab axis in mouse. Some studies have shown that the orientation of first cleavage division in mouse embryos can predict the orientation of the future Em–Ab axis in the blastocyst (Gardner, Reference Gardner2001; Piotrowska & Zernicka-Goetz, Reference Piotrowska and Zernicka-Goetz2001; Piotrowska et al., Reference Piotrowska, Wianny, Pedersen and Zernicka-Goetz2001; Zernicka-Goetz, Reference Zernicka-Goetz2002; Fujimori et al., Reference Fujimori, Kurotaki, Miyazaki and Nabeshima2003; Plusa et al., Reference Plusa, Hadjantonakis, Gray, Piotrowska-Nitsche, Jedrusik, Papaioannou, Glover and Zernicka-Goetz2005). Other studies have shown that the Em–Ab axis of mouse embryos is formed relatively late (Alarcon & Marikawa, Reference Alarcon and Marikawa2003; Motosugi et al., Reference Motosugi, Bauer, Polanski, Solter and Hiiragi2005; Waksmundzka et al., Reference Waksmundzka, Wisniewska and Maleszewski2006; Kurotaki et al., Reference Kurotaki, Hatta, Nakao, Nabeshima and Fujimori2007; Ohsugi et al., Reference Ohsugi, Zheng, Baibakov, Li and Dean2008).
Some studies have reported that there are two alternatives ways for fission in 2-cell development to the 4-cell stage. One alternative is meridional division (M-division), in which the cleavage plane is parallel to the animal–vegetal pole axis (A–V axis), the other alternative is equatorial/oblique division (E-division), in which the spindle is rotated at roughly 90° before or during cytokinesis, and the cleavage plane is perpendicular or oblique to the A–V axis (Gulyas, Reference Gulyas1975; Gardner, Reference Gardner2002). Studies have clarified a relationship between the orientation and the order of blastomere division from the 2- to the 4-cell stage, and the orientation of the Em–Ab axis (Gardner, Reference Gardner2001; Piotrowska & Zernicka-Goetz, Reference Piotrowska and Zernicka-Goetz2001, Reference Piotrowska and Zernicka-Goetz2002; Piotrowska et al., Reference Piotrowska, Wianny, Pedersen and Zernicka-Goetz2001; Zernicka-Goetz, Reference Zernicka-Goetz2002; Fujimori et al., Reference Fujimori, Kurotaki, Miyazaki and Nabeshima2003; Plusa et al., Reference Plusa, Hadjantonakis, Gray, Piotrowska-Nitsche, Jedrusik, Papaioannou, Glover and Zernicka-Goetz2005). When the first division of the 2-cell blastomere is the M-division and the latter is the E-division, the progeny of the first cleavage blastomere contributes predominantly to the embryonic part of the blastocyst (Piotrowska-Nitsche & Zernicka-Goetz, Reference Piotrowska-Nitsche and Zernicka-Goetz2005; Piotrowska-Nitsche et al., Reference Piotrowska-Nitsche, Perea-Gomez, Haraguchi and Zernicka-Goetz2005). Furthermore, if the zygotic genome cannot be activated at the 1-cell stage, embryonic development would be blocked at the second mitosis (Bolton et al., Reference Bolton, Oades and Johnson1984). This situation means that the order and orientation of the blastomere cleavage division in the second mitosis and the specialization of the Em–Ab axis in the 2-cell blastomeres may be regulated by zygotic genes. The asymmetric distribution of zygotic gene products in 2-cell blastomeres influences the specialization of the Em–Ab axis directly.
Axin1, Cdc25cx and Cdkn2d are activated in 2-cell mouse embryos (Zeng & Schultz, Reference Zeng and Schultz2005). Axin1 is an inhibitor of Wnt that signals and regulates an early step in embryonic-axis formation in mammals and amphibians (Zeng et al., Reference Zeng, Fagotto, Zhang, Hsu, Vasicek, Perry, Lee, Tilghman, Gumbiner and Costantini1997). Therefore, we inferred that the asymmetrical distribution of Axin1 would be related to the polarity of the Em–Ab axis.
It is well known that Cdc25c and Cdkn2d are related to cell mitosis, which may have an effect on the orientation and the order of the cell cleavage. The dissymmetrical distribution of Axin1/Cdc25c/Cdkn2d mRNA in 2-cell blastomeres has not been reported. Our work, therefore focusses on the link between the dissymmetric distribution of Axin1/Cdc25c/Cdkn2d in 2-cell blastomeres and the formation of the Em–Ab axis to determine if different blastomeres have different fates at the 2-cell stage.
Materials and methods
Animals
C57BL/6 and CBA mice were purchased from the Animal Centre of Xi'an Jiaotong University (Xi'an, China). Mice were given free access to food and water and bred under controlled conditions (12 h of light, 12 h of darkness; temperature: 20–22°C).
Collection of two blastomeres in 2-cell embryos
F1 (C57BL/6 × CBA) females were superovulated by intraperitoneal injection of 10 IU of pregnant mares serum gonadotrophin (PMSG, Ningbo, China) and intraperitoneal injection of 10 IU of human chorionic gonadotrophin (hCG, Ningbo, China) 48 h later, then they were mated with male mice. Subsequently, 2-cell embryos were collected at 46–48 h post-hCG (late 2-cell stage) and digested with 0.25% Pronase. After the zona pellucida had disappeared, the embryos were placed into FHM medium that contained 4 mg/ml of bovine serum albumin (BSA) and amino acids, and then bisection was performed. Pairs of blastomeres of intact morphology were washed twice with diethylpyrocarbonate–phosphate-buffered saline (DEPC–PBS). Then single blastomere (in 1 μl DEPC–PBS) was put into a 0.2 ml polymerase chain reaction (PCR) tube, respectively, and added with 10 μl resuspension buffer and 1 μl lysis buffer (Invitrogen, 11739–010), and subjected to cDNA synthesis and quantitative PCR (qPCR) detection.
Synthesis of cDNA
The single blastomere cDNA was synthesized with Cells-Direct cDNA Synthesis System with a qPCR kit (Invitrogen) according to manufacturer's instruction. In brief, a single blastomere was lysed in lysis buffer; DNase I and DNase I buffer were added to digest genomic DNA; then the RT-Reaction Mix and Enzyme Mixture were used to generate double-stranded cDNA and mRNA; eventually single-stranded cDNA was obtained by digestion with RNase H and this cDNA was stored at −20°C for subsequent PCR and real-time PCR analysis.
Quantitative analysis of Axin1, Cdc25c and Cdkn2d mRNA
Specific primers for the internal standard β-actin (146-bp products), gene Axin1 (142-bp products), gene Cdc25c (248-bp products) and Cdkn2d (161-bp products) were designed using Primer 5.0 software (Applied Biosystems) based on the GenBank database. The β-actin primers (GenBank accession no. NM_007393) were: sense primer 5′-CCCATCTACGAGGGCTAT-3′ and antisense primer 5′-ATGTCACGCACGATTTCC-3′; the Axin1 primers (GenBank accession no. NM_009733.1) were: sense primer 5′-CCTCTACCTCACATTCCTCGCACTT-3′ and antisense primer 5′- TCAACCGTTCCTCCAACTTTTCT-3′; the Cdc25c primers (GenBank accession no. NM_009860.2) were: sense primer 5′-GAAGCATCTGAGCAGTCCCATTAC-3′ and antisense primer 5′-CTGGCACCGTTGGCAGCACAC-3′; the Cdkn2d primers (GenBank accession no. NM_009878.2) were: sense primer 5′-TGTTCGGAAGTGTTG-3′ and antisense primer 5′-CAGGGCATTGACATCAGCACC-3′.
Real-time PCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) with Smart Cycler (Cepheid). Each real-time PCR reaction was carried out in 25 μl of reaction mixture containing 12.5 μl Platinum SYBR Green qPCR SuperMix-UDG, 0.5 μl of 0.2 μM sense primer, 0.5 μl of 0.2 μM antisense primer, 4 μl of template and 7.5 μl distilled water. Reaction conditions were as follows: β-actin, 50°C for 2 min (UDG incubation), 95°C for 2 min, followed by 45 cycles of 95°C for 15 s, 58°C for 30 s and 72°C for 10 s; Axin1, 50°C for 2 min (UDG incubation), 95°C for 2 min, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 10 s; Cdc25c, 50°C for 2 min (UDG incubation), 95°C for 2 min, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 17 s; Cdkn2d, 50°C for 2 min (UDG incubation), 95°C for 2 min, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 12 s. For the negative control, distilled water was used as template. After cycling, melting curve analysis was undertaken to verify the amplification of the specific target. Each reaction was performed three times. Standard curves used in this study were established with several dilutions of plasmid including β-actin, Axin1, Cdc25c and Cdkn2d (Fig. 1).
Figure 1 Standard curves obtained with several dilutions of several genes from crossing points (cycler numbers) plotted against the log concentration of the serial dilution. (A) β-actin; (B) Axin1; (C) Cdc25c; (D) Cdkn2d.
Statistical analysis
All experiments were repeated three to five times. Real-time PCR data were analysed by double standard curve method. Data were analysed with the software package SPSS. Difference was considered statistically significant when the p-value was < 0.05.
Results and Discussion
These studies were carried out to investigate the expression patterns of Axin1, Cdc25c and Cdkn2d mRNA in blastomeres of 2-cell mouse embryos. Axin1, Cdc25c and Cdkn2d were expressed differently among blastomeres at the 2-cell stage. When compared levels in the two blastomeres of the 2-cell embryo, the level of Axin1 mRNA was significantly higher in one compared with other (p < 0.05, Fig. 2). The variation trend for Cdc25c (p < 0.05; Fig. 3) and Cdkn2d (p < 0.01; Fig. 4) mRNA expression was similar to that for Axin1. The highest level of expression of three genes was detected in the same blastomere of the 2-cell embryos. We confirmed that the asymmetrical distribution patterns of Axin1, Cdc25c and Cdkn2d transcripts in 2-cell embryos by performing PCR on cDNA from individual blastomeres dissected from 2-cell embryos (Fig. 5).
Figure 2 Axin1 mRNA expression in the two blastomeres of the 2-cell stage mouse embryo. The data are given as fold expression. *p-value < 0.05 indicates a significant difference. The expression of Axin1 is normalized on the basis of β-actin expression.
Figure 3 Cdc25c mRNA expression in the two blastomeres of the 2-cell stage mouse embryo. The data are given as fold expression. *p-value < 0.05 indicates a significant difference. The expression of Cdc25c is normalized on the basis of β-actin expression.
Figure 4 Cdkn2d mRNA expression in the two blastomeres of the 2-cell stage mouse embryo. The data are given as fold expression. *p-value < 0.05 indicates a significant difference. The expression of Cdkn2d is normalized on the basis of β-actin expression.
Figure 5 RT-PCR analysis for β-actin, Axin1, Cdc25c and Cdkn2d mRNA in individual blastomeres of the 2-cell stage mouse embryo. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis for β-actin mRNA in individual 2-cell blastomeres. Lane 1, one blastomere from a 2-cell stage embryo; lane 2, the second blastomere from the same 2-cell stage embryo; lane 3,4,5,6, negative RT control of β-actin, Axin1, Cdc25c and Cdkn2d. (B) Reverse transcription polymerase chain reaction (RT-PCR) analysis for Axin1, Cdc25c and Cdkn2d mRNA in individual 2-cell stage blastomeres. Lanes 1, 2. Axin1 mRNA expression by RT-PCR; Lanes 3, 4. Cdc25c mRNA expression by RT-PCR; Lanes 5, 6, Cdkn2d mRNA expression by RT-PCR. Lanes 1, 3, 5. one blastomere from the 2-cell stage embryo; Lanes 2, 4, 6. the second blastomere from the same 2-cell stage embryo.
Axin1 is an inhibitor of Wnt signaling and negatively regulates the response to an axis-inducing signal. Injection of Axin1 mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signalling through the Wnt pathway (Zeng et al., Reference Zeng, Fagotto, Zhang, Hsu, Vasicek, Perry, Lee, Tilghman, Gumbiner and Costantini1997). In the 2-cell embryo, the highest expression of Axin1 was found in the one blastomere, suggesting that the asymmetrical distribution of the Axin1 is related to the polarity of the Em–Ab axis.
The Cdc25c phosphatase is one of the three members of the Cdc25 protein family in mammalian cells which is responsible for the dephosphorylation and the activation of cyclin-dependent kinases (CDK)–cyclin complexes at the key transitions of the cell cycle (Sadhu et al., Reference Sadhu, Reed, Richardson and Russell1990; Galaktionov & Beach, Reference Galaktionov and Beach1991; Nagata et al., Reference Nagata, Igarashi, Jinno, Suto and Okayama1991). Cdc25c was found to be active during late G2 and mitosis (Hoffmann & Karsenti, Reference Hoffmann and Karsenti1994), and plays significant roles in the regulation of the activity of the mitosis promoting factor cdk1–cyclinB by dephosphorylation, thus triggering G2/M transition and the progression into mitosis (Morris et al., Reference Morris, Heitz, Mery, Heitz and Divita2000). Indeed, microinjection of cells with anti-Cdc25c antibodies, or transfection with inactive Cdc25c mutants, prevents entry into mitosis (Millar et al., Reference Millar, Blevitt, Gerace, Sadhu, Featherstone and Russell1991; Lammer et al., Reference Lammer, Wagerer, Saffrich, Mertens, Ansorge and Hoffmann1998). It is well known that there is non-synchronization of cell division in embryos. Our study showed that one blastomere had highest Cdc25c expression in the 2-cell stage, which suggested that this blastomere entered mitosis through the G2/M checkpoint in advance.
Cdkn2d (p19, INK4d) is a member of the INK4 family of cyclin-dependent kinase inhibitors. This protein has been shown to form a stable complex with CDK4 or CDK6, and prevent the activation of the CDK kinases, thus it regulates progression through the G1 phase of the cell cycle. The abundance of the transcript of Cdkn2d was found to oscillate in a cell-cycle-dependent manner with the lowest expression at mid G1 and a maximal expression during S phase (Okuda et al., Reference Okuda, Hirai, Valentine, Shurtleff, Kidd, Lahti, Sherr and Downing1995). Our results show that the Cdkn2d gene was highly expressed in the one blastomere, suggesting that this blastomere moves into the S phase in advance.
Wnt signalling is well known to promote cell cycle progression (Davidson & Niehrs, Reference Davidson and Niehrs2010). Wnt signalling plays critical roles in cell proliferation, and is regulated by various proteins, including Axin1 (Salahshor & Woodgett, Reference Salahshor and Woodgett2005). Another report has demonstrated that Axin1 over-expression facilitated multinuclear giant cell formation in the gastric cancer cell line AGS (Kim et al., Reference Kim, Kim, Ryu, Jho, Song, Jang and Kee2005). In addition, several studies have indicated that Axin1 and Cdc25c localize to centrosomes and regulate the mitotic spindle apparatus (Busch et al., Reference Busch, Barton, Morgenstern, Gotz, Gunther, Noll and Montenarh2007; Fumoto et al., Reference Fumoto, Kadono, Izumi and Kikuchi2009; Kim et al., Reference Kim, Choi, Song, Kim, Seo, Jho and Kee2009). This situation indicates that the orientation and order of cell division are regulated by the Wnt pathway (Segalen & Bellaiche, Reference Segalen and Bellaiche2009). Our study of the distribution of Axin1, Cdc25c and Cdkn2d mRNA in individual 2-cell blastomeres by real-time PCR and RT-PCR revealed that the highest level expression patterns of three genes were detected in the same blastomere of the 2-cell embryos, and further supported the idea that the cell cycle and Wnt signalling are also linked directly to early embryo development. This link will affect directly the orientation and order of blastomere division from the 2- to the 4-cell stage.
In summary, our data demonstrated that embryonic asymmetry had already appeared at the 2-cell stage and these differentially expressed genes may affect differentiation in embryo development. Further study is needed to explore the mechanism by which Axin1, Cdc25c and Cdkn2d affect the orientation and order of blastomere division from the 2- to the 4-cell stage.
Acknowledgements
This work was supported by grants from National Programs for High Technology Research and Development of China (863 program) (2001AA213081). We thank Hongbing Li for providing the technology for the real-time quantitative PCR used in this study, Yongsheng Wang, Yulong He, Yuehong Wu, Shengli Mi, Zhengyuan Su, Yanfei Yang for helpful suggestions and discussions in preparing the manuscript.
