PDGFRβ Activation Induced the Bovine Embryonic Genome Activation via Enhanced NFYA Nuclear Localization

Abstract

Embryonic genome activation (EGA) is a critical step during embryonic development. Several transcription factors have been identified that play major roles in initiating EGA; however, this gradual and complex mechanism still needs to be explored. In this study, we investigated the role of nuclear transcription factor Y subunit A (NFYA) in bovine EGA and bovine embryonic development and its relationship with the platelet-derived growth factor receptor-β (PDGFRβ) by using a potent selective activator (PDGF-BB) and inhibitor (CP-673451) of PDGF receptors. Activation and inhibition of PDGFRβ using PDGF-BB and CP-673451 revealed that NFYA expression is significantly (p < 0.05) affected by the PDGFRβ. In addition, PDGFRβ mRNA expression was significantly increased (p < 0.05) in the activator group and significantly decreased (p < 0.05) in the inhibitor group when compared with PDGFRα. Downregulation of NFYA following PDGFRβ inhibition was associated with the expression of critical EGA-related genes, bovine embryo development rate, and implantation potential. Moreover, ROS and mitochondrial apoptosis levels and expression of pluripotency-related markers necessary for inner cell mass development were also significantly (p < 0.05) affected by the downregulation of NFYA while interrupting trophoblast cell (CDX2) differentiation. In conclusion, the PDGFRβ-NFYA axis is critical for bovine embryonic genome activation and embryonic development.

Keywords: NFYA; PDGFRβ; activator; bovine blastocysts; embryonic genome activation.

Figure 1

Protein expression and mRNA expression of NFYA, PDGFRβ, and PDGFRα during bovine embryonic development. (A) Immunofluorescent expression of NFYA, PDGFRβ, and PDGFRα from COCs to day-8 blastocyst stage. (B) Relative mRNA expression of NFYA, PDGFRβ, and PDGFRα from MII to day-8 blastocyst stage. Data are presented as the mean ± SEM. For immunofluorescent staining, COCs/MII stage oocytes/zygotes/two cell/four cell/8-cell/16-cell-stage embryos and day 8 blastocysts, 20 per group, were used. For mRNA expression analysis, MII stage oocytes/zygotes/two-cell/four-cell-stage embryos, 20 per group; 8-cell and 16-cell embryos, 15 per group, and 6 blastocysts per group were used in triplicates. The experiments were repeated three times. Scale bar: 20 µm. ns = not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 denote significant differences; SEM—standard error of mean. BF = Bright Field.

Figure 2

PDGFRβ activation and inhibition effects on NFYA. (A) Immunofluorescent expression of PDGFRβ and NFYA in 8-cell embryos, 16-cell embryos, and day-8 bovine blastocysts control, activator, and inhibitor groups. (B) Relative PDGFRβ, NFYA, and PDGFRα gene expression through RT-qPCR in 8-cell embryos, 16-cell embryos, and day-8 bovine blastocysts of control, activator, and inhibitor groups. (C) Relative mRNA expression of NFYB and NFYC in 8-cell embryos, 16-cell embryos, and day-8 blastocyst control, activator, and inhibitor groups. (D) Dose-dependent response of PDGF-BB and CP-673451 on embryo cleavage percentage and blastocyst developmental percentage. Data are presented as the mean ± SEM. For mRNA analysis, 8-cell and 16-cell embryos, 15 per group, and 6 blastocysts per group were used in triplicates. For immunofluorescent staining, n = 20 8-cell and 16-cell embryos and n = 15 blastocysts per group were used. The experiments were repeated three times. Scale bar: 20 µm. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 denote significant differences. SEM—standard error of mean.

Figure 2

PDGFRβ activation and inhibition effects on NFYA. (A) Immunofluorescent expression of PDGFRβ and NFYA in 8-cell embryos, 16-cell embryos, and day-8 bovine blastocysts control, activator, and inhibitor groups. (B) Relative PDGFRβ, NFYA, and PDGFRα gene expression through RT-qPCR in 8-cell embryos, 16-cell embryos, and day-8 bovine blastocysts of control, activator, and inhibitor groups. (C) Relative mRNA expression of NFYB and NFYC in 8-cell embryos, 16-cell embryos, and day-8 blastocyst control, activator, and inhibitor groups. (D) Dose-dependent response of PDGF-BB and CP-673451 on embryo cleavage percentage and blastocyst developmental percentage. Data are presented as the mean ± SEM. For mRNA analysis, 8-cell and 16-cell embryos, 15 per group, and 6 blastocysts per group were used in triplicates. For immunofluorescent staining, n = 20 8-cell and 16-cell embryos and n = 15 blastocysts per group were used. The experiments were repeated three times. Scale bar: 20 µm. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 denote significant differences. SEM—standard error of mean.

Figure 3

PDGFRβ and NFYA have been linked to zygotic genome activation. (A) Relative mRNA expression of zygotic genome activation related to DUXA, GSC, ARGFX, SP1, and DPRX genes in the control, activator, and inhibitor groups. (B) Relative mRNA expression of SRF and ZXH1 in control, activator, and inhibitor groups. (C) Relative mRNA expression levels of trophoblast-related CDX2. (D) Relative mRNA expression levels of OCT4, NANOG, SOX2, KLF4, and SALL4 pluripotency-related genes. Data are shown as the mean ± SEM. For mRNA analysis, 8-cell and 16-cell embryos, 15 per group, and 6 blastocysts per group were used in triplicates. ns = not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 denote significant differences. SEM—standard error of mean.

Figure 4

NFYA nuclear localization via PDGFRβ activation. (A). Relative mRNA expression of MAPK1 and AKT in control, activator, and inhibitor groups. (B). Relative mRNA expression of JAK2 and STAT3. (C) Relative mRNA expression of CIITA, CREB in control, activator, and inhibitor groups. For mRNA analysis, 8-cell and 16-cell embryos, 15 per group, and 6 blastocysts per group were used in triplicates. The experiments were repeated three times. Data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 denotes significant differences. SEM—standard error of mean.

Figure 5

PDGFRβ and NFYA affect embryonic mitochondria and implantation potential of bovine embryos. (A) JC-1 staining of day-8 blastocyst control, activator, and inhibitor groups (n = 15 blastocysts per group). The scale bar indicates 20 µm. (B) H₂DCFDA staining in embryos in control, activator, and inhibitor groups. The scale bar indicates 100 µm. (C) The TUNEL assay in day-8 blastocysts to examine apoptosis in control, activator, and inhibitor groups (n = 15 blastocysts per group). The scale bar indicates 20 µm. (D) The invasion area and cell proliferation of implanted blastocysts in the control, activator, and inhibitor groups (three blastocysts per group). The magnification is 40×. All the experiments were repeated three times. Data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.01 denote significant differences. SEM—standard error of mean. BF = Bright Field.