Fig 1.
NPB volume and development of extra-NPB-injected embryos.
(A) NPB volume and single NPB formation in pronuclei of extra-NPB-injected ICSI (ICSI+NPB) embryos. The total volumes of NPBs were analyzed in ICSI+Sham (n = 18) and ICSI+NPB (n = 61) zygotes. Bars represent the mean ± SEM. Two-tailed, unpaired Student’s t-test was performed. The percentages of single NPB formation in pronucleus were analyzed in ICSI+Sham (n = 378) and ICSI+NPB (n = 494) pronuclei. Chi-square test was performed. ***P<0.001: not significant (N.S.). Arrowheads indicate NPBs in pronuclei. The scale bar is 50 μm. (B) In vitro and in vivo development of ICSI+NPB embryos. The rates of embryos that formed pronuclei and developed to the two-cell stage (ICSI+Sham: n = 221; ICSI+NPB: n = 213), and development to the morula/blastocyst stage (ICSI+ Sham: n = 99; ICSI+NPB: n = 104) were analyzed. The rates of full-term development were analyzed after the transfer of two-cell-stage embryos into surrogate mothers (ICSI+Sham: n = 105/8 and ICSI+NPB: 81/7; embryos/recipients). Chi-square test was performed. not significant (N.S.). The scale bar is 50 μm. (C) Cell numbers and cell types of blastocysts were compared between ICSI+Sham embryos (n = 46) and ICSI+NPB embryos (n = 52). Cell numbers of the trophectoderm (TE) and inner cell mass (ICM) were counted after immunostaining for Cdx2 (green) and Oct3/4 (red), respectively. DAPI staining marks chromatin in blue. Cdx2/Oct3/4 doubly-positive cells (Transition) were counted on merged images. Metaphase cells (DAPI staining only) were counted on triple-merged images. The scale bar is 20 μm. Error bars in the graph indicate the standard error of the mean. Two-tailed, unpaired Student’s t-test was performed.
Fig 2.
NPB volume affects the number of NPBs in the pronucleus.
(A) Experimental scheme. Oocytes were injected with an extra-MII spindle and then subjected to parthenogenetic activation (Parthenogenetic+MII spindle). An extra-NPB was injected into the cytoplasm of MII oocytes before extra-MII-spindle injection (Parthenogenetic+MII spindle+NPB). Polar body extrusion was prevented by latrunculin A treatment. The volume of NPB per pronucleus was doubled in Haploid parthenogenetic embryos and halved in Parthenogenetic+MII spindle embryos, whereas each pronucleus had a haploid number of chromosomes. (B) Histone H3 Immunostaining and counting of NPBs in Parthenogenetic+MII spindle+NPB embryos. Embryos were fixed at 10 h after ICSI or parthenogenetic activation for immunostaining with histone H3 (green). Images of Parthenogenetic+MII spindle and Parthenogenetic+MII spindle+NPB embryos were combined with 2–3 different single Z slice images in the same embryo. The scale bar is 10 μm. (C) Single NPB formation in embryos with different NPB/pronucleus ratios. The percentages of pronuclei with single NPB formation were analyzed in ICSI (n = 110), Diploid parthenogenetic (n = 44), Haploid parthenogenetic (n = 34), Parthenogenetic+MII spindle (n = 108) and Parthenogenetic+MII spindle+NPB (n = 47) pronuclei. Chi-square test was performed. *P<0.05, **P<0.01, ***P<0.001. (D) Total NPB volumes in different NPB/pronucleus ratio embryos. The total volumes of NPBs were analyzed in ICSI (n = 13), Diploid parthenogenetic (n = 42), Haploid parthenogenetic (n = 36), Parthenogenetic+MII spindle (n = 17) and Parthenogenetic+MII spindle+NPB embryos (n = 9). Bars represent the mean ± SEM. Two-tailed, unpaired Student’s t-test was performed. ***P<0.001: not significant (N.S.).
Fig 3.
NPB formation in SCNT embryos.
(A) NPB volume and single NPB formation in extra-NPB-injected SCNT embryos (SCNT+NPB). The total volumes of NPBs were analyzed in sham-operated SCNT embryos (SCNT+Sham: n = 67) and SCNT+NPB embryos (n = 56). Bars represent the mean ± SEM. Two-tailed, unpaired Student’s t-test was performed. The percentages of pseudo-pronuclei with single NPB formation were analyzed in SCNT+Sham embryos (n = 253 pronuclei) and SCNT+NPB embryos (n = 356 pronuclei). Chi-square test was performed. ***P<0.001. Arrowheads indicate NPBs in pseudo-pronuclei. The scale bar is 50 μm. (B) Time-lapse images of NPB formation in the extra-NPB-injected SCNT embryos. Live-cell imaging was performed after injection with EGFP-NPM2 (green) and H2B-mCherry (red) mRNA into oocytes at MII followed by the NPB injection and SCNT. Time after start of imaging (h:mm). The scale bar is 20 μm.
Fig 4.
Development of SCNT embryos injected with extra-NPBs.
(A) In vitro and in vivo development of SCNT embryos injected with extra-NPBs (SCNT+NPB). The rates of embryos that formed pseudo-pronuclei, developed to the two-cell stage (SCNT+Sham: n = 432 embryos; SCNT+NPB: n = 467 embryos), and developed to the morula/blastocyst stage (SCNT+Sham: n = 123 embryos; SCNT+NPB: n = 108 embryos) were analyzed. The rate of full-term development was analyzed after the transfer of two-cell-stage embryos into surrogate mothers (SCNT+Sham: n = 189/19 embryos/recipients; SCNT+NPB: n = 178/14 embryos/recipients). Chi-square test was performed. not significant (N.S.). ***P<0.001. The scale bar is 50 μm. (B) Cell numbers and cell types of blastocysts were compared between SCNT+Sham (n = 51) and SCNT+NPB embryos (n = 40). Cell numbers of the trophectoderm (TE) and inner cell mass (ICM) were counted after immunostaining for Cdx2 (green) and Oct3/4 (red), respectively. DAPI staining marks chromatin in blue. Cdx2/Oct3/4 doubly positive cells (Transition) were counted on merged images. Metaphase cells (DAPI staining only) were counted on triple-merged images. The scale bar is 20 μm. Error Bars in the graph indicate the standard error of the mean. Two-tailed, unpaired Student’s t-test was performed. (C) Cloned offspring with the placenta (Left) and adult cloned offspring originating (Center) from SCNT+NPB embryos using the same donor cells. An adult offspring from SCNT+NPB embryos with full fertility is also shown (Right).