De novo formation of nucleoli in developing mouse embryos originating from enucleolated zygotes

Abstract

The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials.

Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

“…Indeed, maternal nucleoli are essential for successful early embryonic development [26]. The nucleoli in developing embryos could also be formed by de novo synthesized materials [27]. Nucleoli are closely related to embryonic genome activation (EGA).…”

Nucleologenesis and Nucleolotransfer in Mammalian Oocytes: A Review

“…According to our experiences, for enucleation, the M2 medium (Zenith Biotech ZFM2-050; Czech Republic) supplemented with PVA (Sigma P8136; Czech Republic) is superior to M2 medium (Zenith Biotech ZFM2-050; Czech Republic) supplemented with BSA (Sigma A3311; Czech Republic), as the cytoplasmic membrane appears to be more compact. The enucleolation from zygotes is similar to the method reported for the oocytes (8). A, B): The first step of this process is the microsurgical removal of nucleoli from fully grown oocytes as nucleoloplasts.…”

“…It has been commonly accepted that oocyte and zygote NLB/ NPB serve as repository of material from which differentiated nucleoli in embryos are gradually formed during embryogenesis (5,6). Recent observations have challenged this developmental biology dogma and showed that they are important only during a very short time period after fertilization (7,8).…”

“…During pronuclear development, these nucleoli fuse and form a single prominent structure (8). As it has been commonly accepted until recently that this structure is gradually transformed during subsequent embryonic development into typical tripartite nucleoli, it was also termed as so called "nucleolus precursor bodies -NPBs".…”

“…(D): When NPBs were removed from late zygotes, no NPBs were detected in pronuclei or nuclei of enucleolated zygotes or in two-cell embryos. However, later (approximately at the four-cell stage) these embryos formed nucleoli de novo and the embryos have developed into the blastocyst stage after parthenogenetic activation or IVF (8). (E): If NLBs were removed from oocytes and introduced back into more advanced parthenogenetic or IVF embryos (originated from enucleolated oocytes, i.e.…”