Hirohisa Kyogoku

Large Cytoplasm Is Linked to the Error-Prone Nature of Oocytes

Chromosome segregation during meiosis in oocytes is error prone. The uniquely large cytoplasmic size of oocytes, which provides support for embryogenesis after fertilization, might be a predisposing factor for meiotic errors. However, this hypothesis remains unproven. Here, we show that cytoplasmic size affects the functionality of the acentrosomal spindle. Artificially decreasing the cytoplasmic size in mouse oocytes allows the acentrosomal spindle poles to have a better-focused distribution of microtubule-organizing centers and to biorient chromosomes more efficiently, whereas enlargement of the cytoplasmic size has the opposite effects. Moreover, we found that the cytoplasmic size-dependent dilution of nuclear factors, including anaphase inhibitors that are preformed at the nuclear membrane, limits the spindles capacity to prevent anaphase entry with misaligned chromosomes. The present study defines a large cytoplasmic volume as a cell-intrinsic feature linked to the error-prone nature of oocytes. This may represent a trade-off between meiotic fidelity and post-fertilization developmental competence.

Nucleolus Precursor Body (NPB): A Distinct Structure in Mammalian Oocytes and Zygotes

Nucleoli in mammalian oocytes and zygotes, sometimes referred to as nucleolus precursor bodies (NPBs), are compact and morphologically different from nucleoli in somatic cells. We applied a unique NPB analyzing method “enucleolation” technique to zygotes to remove the NPBs. It has been reported that oocyte NPBs are essential for embryonic development; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygotes and embryos, leading to developmental failure. However, we found that when NPBs were removed from zygotes, the zygotes developed successfully to live-born pups. These results indicated that oocyte NPBs are essential for embryonic development, but zygote NPBs are not. In addition, the enucleolated zygotes formed somatic-type nucleoli during early embryonic development, demonstrating that somatic-type nucleoli do not originate from zygote NPBs. We summarize our recent investigation on NPBs, and provide additional comments and findings.

Nucleoli From Growing Oocytes Inhibit the Maturation of Enucleolated, Full-Grown Oocytes in the Pig

In mammals, the nucleolus of full‐grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full‐grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non‐treated or actinomycin D‐treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re‐injection of nucleoli from growing oocytes (23%), but not when nucleoli from full‐grown oocytes were re‐injected into enucleolated, growing oocytes (49%). When enucleolated, full‐grown oocytes were injected with nucleoli from growing or full‐grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full‐grown oocytes injected with nucleoli from full‐grown oocytes matured to metaphase II (56%), whereas injection with growing‐oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing‐oocyte nucleolus is involved in the oocytes meiotic arrest, and that the full‐grown oocyte nucleolus has lost the ability. Mol. Reprod. Dev. 78:426–435, 2011.

Nucleoli from growing oocytes support the development of enucleolated full‐grown oocytes in the pig

Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full‐grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full‐grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 µm in diameter) and full‐grown porcine oocytes (120 µm) were collected from small (0.6–1.0 mm) and large antral follicles (4–5 mm), respectively. The nucleolus was aspirated from full‐grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full‐grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full‐grown oocytes. After activation by electro‐stimulation, nucleoli were formed in pronuclei of sham‐operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full‐grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full‐grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full‐grown oocytes during early embryonic development. Mol. Reprod. Dev. 77: 167–173, 2010.

Nucleoli from Two-Cell Embryos Support the Development of Enucleolated Germinal Vesicle Oocytes in the Pig

ABSTRACT Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Can Nucleoli Be Markers of Developmental Potential in Human Zygotes

In 1999, Tesarik and Greco reported that they could predict the developmental potential of human zygotes from a single static evaluation of their pronuclei. This was based on the distribution and number of specific nuclear organelles - the nucleoli. Recent studies in mice show that nucleoli play a key role in parental genome restructuring after fertilization, and that interfering with this process may lead to developmental failure. These studies thus support the Tesarik-Greco evaluation as a potentially useful method for selecting high-quality embryos in human assisted reproductive technologies. In this opinion article we discuss recent evidence linking nucleoli to parental genome reprogramming, and ask whether nucleoli can mirror or be used as representative markers of embryonic parameters such as chromosome content or DNA fragmentation.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

Single nucleolus precursor body formation in the pronucleus of mouse zygotes and SCNT embryos

Mammalian oocytes and zygotes have nucleoli that are transcriptionally inactive and structurally distinct from nucleoli in somatic cells. These nucleoli have been termed nucleolus precursor bodies (NPBs). Recent research has shown that NPBs are important for embryonic development, but they are only required during pronuclear formation. After fertilization, multiple small NPBs are transiently formed in male and female pronuclei and then fuse into a single large NPB in zygotes. In cloned embryos produced by somatic cell nuclear transfer (SCNT), multiple NPBs are formed and maintained in the pseudo-pronucleus, and this is considered an abnormality of the cloned embryos. Despite this difference between SCNT and normal embryos, it is unclear how the size and number of NPBs in pronuclei is determined. Here, we show that in mouse embryos, the volume of NPB materials plays a major role in the NPB scaling through a limiting component mechanism and determines whether a single or multiple NPBs will form in the pronucleus. Extra NPB- and extra MII spindle-injection experiments demonstrated that the total volume of NPBs was maintained regardless of the pronucleus number and the ratio of pronucleus/NPB is important for fusion into a single NPB. Based on these results, we examined whether extra-NPB injection rescued multiple NPB maintenance in SCNT embryos. When extra-NPBs were injected into enucleated-MII oocytes before SCNT, the number of NPBs in pseudo-pronuclei of SCNT embryos was reduced. These results indicate that multiple NPB maintenance in SCNT embryos is caused by insufficient volume of NPB.

Cytoplasmic removal, enucleation, and cell fusion of mouse oocytes

Meiotic divisions in females occur in fully grown oocytes that have a large cytoplasmic volume. The intracellular processes that are needed to accomplish meiotic divisions, such as spindle formation, chromosome segregation, and polar body extrusion, are controlled by the concerted actions of nuclear and cytoplasmic factors, which exhibit dynamic quantitative and spatiotemporal changes during meiotic maturation. Thus, distinguishing between meiotic controls that are mediated by cytoplasmic factors and those mediated by nuclear factors helps in the understanding of the mechanisms underlying meiotic divisions. Here, we describe a method to artificially modify the number of nuclei and the volume of the cytoplasm of mouse oocytes through cytoplasmic removal, enucleation, and cell fusion. The oocytes generated by this method are viable and undergo reproducible meiotic divisions exhibiting the effects of altered amounts of cytoplasmic and nuclear factors, which can be analyzed by various assays, such as live imaging microscopy.