Figure 1.
Experimental design for distinguishing the allelic origin of Oct4 mRNA in SCNT and fertilized embryos.
(A) Enucleated C3H/HeN (for brevity, C3H) oocytes were transplanted with C57Bl/6J (for brevity, B6) cumulus cell nuclei to produce SCNT embryos, which were cultured in the presence of aphidicolin (Aph) or in normal medium, resulting in G1/S phase arrested pronuclear oocytes or in cleavage stages, respectively. Fertilized embryos were produced with intact C3H oocytes and B6 sperm and cultured the same way as the SCNT embryos. hpa: hours past activation. (B) Oct4 transcript from the C3H and B6 mouse strains can be distinguished by a restriction-enzyme-sensitive single nucleotide polymorphism (SNP). Oct4 transcript from C3H remains uncut (565 bp), while Oct4 transcript from B6 is cut (429 bp and 136 bp). If found positive for β-actin mRNA, embryos were then processed and analyzed individually.
Figure 2.
Aphidicolin effectively and selectively inhibits DNA synthesis in fertilized embryos.
In mouse development the first round of DNA replication occurs at the pronuclear stage and the major wave of embryonic genome activation occurs at the 2-cell stage. While the deoxyribonucleotide analogue 5-ethynyl-2′-deoxyuridine (EdU) was incorporated into newly synthesized DNA of aphidicolin-negative (Aph−) pronuclear oocytes, no EdU incorporation was detected in Aph+ pronuclear oocytes that were treated with 2 µg/ml Aph for 6 hours (A, A’). When 2-cell embryos in G1 phase were cultured with the ribonucleotide analogue 5-ethynyl uridine (EU), EU was incorporated into newly synthesized mRNA regardless of the 6-hour Aph treatment (B, B’). When Aph treatment was protracted after 6 hours, EU incorporation was unchanged after 24 hours (C, C’) and was reduced after 60 hours (D, D’). Incorporation of EdU and EU was revealed using Click-iT imaging technology. Fluorescence images were taken on a Nikon TE2000 microscope fitted with an UltraView RS3 confocal module, and the fluorescence signals indicative of EdU and EU incorporation were quantified using ImageJ. Scale bar 40 µm. Statistical significance was calculated using t-test with p≤0.05.
Figure 3.
Venn diagram of transcripts elevated ≥2-fold in embryos treated or not treated with aphidicolin.
Pronuclear oocytes were cultured in the presence of aphidicholin (Aph) from 6 to 96 hours post activation (hpa), and compared with sibling embryos that were not treated so as to appreciate how many mRNAs are upregulated regardless of the cell cycle progression. (A) Venn diagram showing mRNAs that accumulate after SCNT when the first round of embryonic DNA replication is suppressed (Aph+) in comparison to mRNAs that accumulate when the embryo can cycle normally (Aph−). (B) Venn diagram showing mRNAs that accumulate after fertilization when the first round of embryonic DNA replication is suppressed (Aph+) in comparison to mRNAs that accumulate when the embryo can cycle normally (Aph−). In both SCNT and fertilization, mRNAs were considered whose abundance increased at least 2-fold compared to MII oocytes.
Figure 4.
Representative images of individual embryos analyzed by allele-specific PCR for Oct4 and Nanog mRNA.
All of the embryos subjected to Oct4 (A) and Nanog (B) analysis were previously found positive for β-actin mRNA. Gel pictures show 7 embryos analyzed for Oct4 and 7 embryos analyzed for Nanog. (A) Oct4 transcripts from 3 Aph+ embryos are only maternal (C3H/HeN amplicon, single band uncut), whereas Oct4 transcript from 4 Aph− embryos are either maternal (no reprogramming) or somatic (C57Bl/6J reprogrammed, cut) or paternal (C57Bl/6J sperm, cut). (B) Aph+ embryos have no Nanog transcript whereas Aph− embryos have Nanog transcript from either the C3H/HeN or the C57Bl/6J allele.
Figure 5.
Results of individual embryos analyzed by allele-specific PCR for Oct4 and Nanog mRNA.
(A) Pooled frequencies of Oct4 and (B) Nanog mRNA expression in SCNT or ICSI embryos after treatment with Aph or CB from 6 hpa. (C) Frequencies of Oct4 mRNA expression in SCNT embryos after treatment with Aph or CB from 6 or 24 hpa. (D) Frequencies of Oct4 mRNA expression in ICSI embryos after treatment with Aph or CB from 6 or 24 hpa. (E) Frequencies of Nanog mRNA expression in SCNT embryos after treatment with Aph or CB from 6 or 24 hpa. (F) Frequencies of Nanog mRNA expression in ICSI embryos after treatment with Aph or CB from 6 or 24 hpa. All of the abovementioned embryos tested positive for β-actin mRNA. Statistical significance was calculated using chi-square test with p≤0.05.
Table 1.
PCR detection of Oct4 mRNA in SCNT and ICSI embryos (C3HxB6).
Figure 6.
Representative images of Aphidicolin (Aph) or cytochalasin B (CB) treated embryos derived from Oct4-GFP nuclei.
Aph−: control embryos not treated with Aph cleave as expected and express OCT4-GFP. Aph+: embryos treated with 2 µg/mL Aph at the 1- or 2-cell stage, which blocks DNA replication and thereby cell division (cytokinesis), fail to express OCT4-GFP. CB: treatment with 5 µg/mL CB prevents cytokinesis without affecting DNA synthesis and OCT4-GFP expression. Scale bar 100 µm.
Table 2.
OCT4-GFP expression in SCNT and ICSI embryos (C3HxOG2).
Table 3.
PCR detection of Nanog mRNA in SCNT and ICSI embryos (C3HxB6).