Figure 1.
Pthlh expression in mouse oocytes and preimplantation embryos.
Quantitative real-time PCR was applied to detect the expression of Pthlh in GV oocytes, MII oocytes, and in embryos at the 1-cell (1-C), 2-cell (2-C), 4-cell (4-C), 8-cell (8-C), morula (MO) and blastocyst (BL) stages. Transcripts levels were normalized against Gapdh expression. Data are presented as means ± SEM.
Figure 2.
The effects of Pthlh siRNA injection on the expression of Pthlh, PTHLH and Pth1r in mouse preimplantation embryos.
A) Quantitative real-time PCR was applied to detect the expression of Pthlh in the Pthlh siRNA-injected ICSI embryos at the 2-cell stage. Transcripts levels were normalized against Gapdh expression. Data are presented as means ± SEM. *p<0.01. B) Pthlh expression level in 2-cell embryos 24 h post-injection. Data are presented as means ± SEM. *p<0.01. C) Pth1r expression level in 2-cell embryos 24 h post-injection. Data are presented as means ± SEM. *p<0.01. D) Western blot results of PTHLH in the Pthlh siRNA-injected group and the control groups 24 h post-injection, GAPDH was used as the loading control.
Table 1.
Effect of Pthlh siRNA on development of mouse ICSI embryos.
Table 2.
Embryonic development following the microinjection of Pthlh siRNA into mouse embryos at the 1-cell stage.
Figure 3.
The effects of Pthlh siRNA injection on the expression of Pthlh (A), Nanog (B) and Pou5f1 (C) in mouse morula stage embryos.
The uninjected embryos and the control siRNA-injected embryos were used as controls. Data are presented as means ± SEM. *p<0.01.
Figure 4.
The effects of Pthlh siRNA injection on OCT4 and NANOG expression in mouse morula stage embryos.
A) Staining pattern of OCT4 in the uninjected (a-a’), the control siRNA-injected (b-b’) and the Pthlh siRNA-injected (c-c’) embryos; B) Staining pattern of NANOG in the uninjected (a-a’), the control siRNA-injected (b-b’) and the Pthlh siRNA-injected (c-c’) embryos; C) Quantification of OCT4/DNA signal intensity in the Pthlh siRNA-injected and the control embryos (n = 14); D) Quantification of NANOG/DNA signal intensity in the Pthlh siRNA-injected and the control embryos (n = 13). OCT4/NANOG, green; DNA, blue. Bar = 20 µm. Data are presented as means ± SEM. *P<0.01.
Figure 5.
The effects of Pthlh siRNA injection on the acetylation of lysine 9 on histone H3 (Ac-H3K9) in mouse preimplantation embryos.
A) The staining pattern of Ac-H3K9 in the uninjected (a-a’ to e-e’), the control siRNA-injected (f-f’ to j-j’) and the Pthlh siRNA-injected (k-k’ to o-o’) embryos at the 2-cell stage (a-a’, f-f’ and k-k’); the 4-cell stage (b-b’, g-g’ and l-l’); the 8-cell stage (c-c’, h-h’ and m-m’); the morula stage (d-d’, i-i’ and n-n’) and the blastocyst stage (e-e’, j-j’ and o-o’). Ac-H3K9, green; DNA, blue. Bar = 20 µm. B) Quantification of Ac-H3K9/DNA signal intensity in the Pthlh siRNA-injected and the control embryos (n = 12). Data are presented as means ± SEM. *P<0.05.
Figure 6.
The effects of Pthlh siRNA injection on the acetylation of H3K14 (Ac-H3K14)
in mouse preimplantation embryos. A) The staining pattern of Ac-H3K14 in the uninjected (a-a’ to e-e’), the control siRNA-injected (f-f’ to j-j’) and the Pthlh siRNA-injected (k-k’ to o-o’) embryos at the 2-cell stage (a-a’, f-f’ and k-k’); the 4-cell stage (b-b’, g-g’ and l-l’); the 8-cell stage (c-c’, h-h’ and m-m’); the morula stage (d-d’, i-i’ and n-n’) and the blastocyst stage (e-e’, j-j’ and o-o’). Ac-H3K14, green; DNA, blue. Bar = 20 µm. B) Quantification of Ac-H3K14/DNA signal intensity in the Pthlh siRNA-injected embryos and the control embryos (n = 12). Data are presented as means ± SEM. *P<0.05, **P<0.01.
Table 3.
Effect of PTHLH (1–141) on development of mouse embryos injected with Pthlh siRNA.
Table 4.
Primer sequences used for real-time PCR.