Figure 1.
Figure 1. Nucleostemin is essential for preimplantation development. (A) X-gal staining of Ns+/− (Ns+/βgeo) preimplantation embryos derived from intercrosses of Ns+/− males with wild-type females. The thick transparent layer surrounding the embryos is the zona pellucida. For A–F, individual embryos or cultures were genotyped by PCR after photomicroscopy. (B) Morphology of E3.5 embryos derived from Ns+/− intercrosses. Embryos were sorted into morphologically normal and abnormal sets. The first set was a mixture of Ns+/+ and Ns+/− genotypes, the second set was exclusively Ns−/−. Although littermate wild-type or heterozygous embryos formed blastocysts with a discernable blastocoel cavity (arrows), ICM (stars), and TE (the outer cell layer surrounding the embryos) at E3.5, Ns−/− embryos resembled morulae and exhibited various degrees of morphological degeneration. (C) In vitro culture demonstrating failure of Ns−/− embryos to develop from compacted morulae to blastocysts. Embryos were collected at E1.5 from Ns+/− intercrosses and cultured individually. Shown are littermate embryos at matched time points. (D) Ns−/− embryos fail to blastulate and hatch in outgrowth cultures. Shown are E3.5 littermate embryos freshly isolated from Ns+/− intercrosses, and after 1–4 d of culture. The Ns−/− embryo shown here was chosen for genotyping because of its degeneration after 48 h of culture. The arrow and arrowhead indicate trophoblast giant cells and ICM-derived cells, respectively. (E) BrdU incorporation assay showing decreased proliferation in Ns−/− embryos. E3.5 embryos were cultured overnight in M16 medium containing BrdU and analyzed. After overnight culture, wild-type embryos (top) developed into late blastocysts with expanded blastocoel and demonstrated extensive proliferation with strong BrdU staining in both the ICM and TE. In contrast, Ns−/− embryos (bottom) failed to develop into blastocysts and incorporated little BrdU. (F) TUNEL staining of embryos indicating increased apoptosis in Ns−/− embryos. Freshly isolated E3.5 embryos were examined by TUNEL staining. Although wild-type embryos (left) showed few labeled cells, Ns−/− embryos demonstrated intense labeling of disorganized blastomeres in partially degenerating embryos (middle), and in most cells of apparently fragmented embryos (right), indicating apoptotic cell death in these mutants. Bars, 40 µm.

Nucleostemin is essential for preimplantation development. (A) X-gal staining of Ns+/− (Ns+/βgeo) preimplantation embryos derived from intercrosses of Ns+/− males with wild-type females. The thick transparent layer surrounding the embryos is the zona pellucida. For A–F, individual embryos or cultures were genotyped by PCR after photomicroscopy. (B) Morphology of E3.5 embryos derived from Ns+/− intercrosses. Embryos were sorted into morphologically normal and abnormal sets. The first set was a mixture of Ns+/+ and Ns+/− genotypes, the second set was exclusively Ns−/−. Although littermate wild-type or heterozygous embryos formed blastocysts with a discernable blastocoel cavity (arrows), ICM (stars), and TE (the outer cell layer surrounding the embryos) at E3.5, Ns−/− embryos resembled morulae and exhibited various degrees of morphological degeneration. (C) In vitro culture demonstrating failure of Ns−/− embryos to develop from compacted morulae to blastocysts. Embryos were collected at E1.5 from Ns+/− intercrosses and cultured individually. Shown are littermate embryos at matched time points. (D) Ns−/− embryos fail to blastulate and hatch in outgrowth cultures. Shown are E3.5 littermate embryos freshly isolated from Ns+/− intercrosses, and after 1–4 d of culture. The Ns−/− embryo shown here was chosen for genotyping because of its degeneration after 48 h of culture. The arrow and arrowhead indicate trophoblast giant cells and ICM-derived cells, respectively. (E) BrdU incorporation assay showing decreased proliferation in Ns−/− embryos. E3.5 embryos were cultured overnight in M16 medium containing BrdU and analyzed. After overnight culture, wild-type embryos (top) developed into late blastocysts with expanded blastocoel and demonstrated extensive proliferation with strong BrdU staining in both the ICM and TE. In contrast, Ns−/− embryos (bottom) failed to develop into blastocysts and incorporated little BrdU. (F) TUNEL staining of embryos indicating increased apoptosis in Ns−/− embryos. Freshly isolated E3.5 embryos were examined by TUNEL staining. Although wild-type embryos (left) showed few labeled cells, Ns−/− embryos demonstrated intense labeling of disorganized blastomeres in partially degenerating embryos (middle), and in most cells of apparently fragmented embryos (right), indicating apoptotic cell death in these mutants. Bars, 40 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal