The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms.

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.

Reconstitution of the oocyte nucleolus in mice through a single nucleolar protein, NPM2.

The mammalian oocyte nucleolus, the most prominent subcellular organelle in the oocyte, is vital in early development, yet its key functions and constituents remain unclear. We show here that the parthenotes/zygotes derived from enucleolated oocytes exhibited abnormal heterochromatin formation around parental pericentromeric DNAs, which led to a significant mitotic delay and frequent chromosome mis-segregation upon the first mitotic division. A proteomic analysis identified nucleoplasmin 2 (NPM2) as a dominant component of the oocyte nucleolus. Consistently, Npm2-deficient oocytes, which lack a normal nucleolar structure, showed chromosome segregation defects similar to those in enucleolated oocytes, suggesting that nucleolar loss, rather than micromanipulation-related damage to the genome, leads to a disorganization of higher-order chromatin structure in pronuclei and frequent chromosome mis-segregation during the first mitosis. Strikingly, expression of NPM2 alone sufficed to reconstitute the nucleolar structure in enucleolated embryos, and rescued their first mitotic division and full-term development. The nucleolus rescue through NPM2 required the pentamer formation and both the N- and C-terminal domains. Our findings demonstrate that the NPM2-based oocyte nucleolus is an essential platform for parental chromatin organization in early embryonic development.

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

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.

Effects of proteasome inhibitors on the nucleolar size of porcine oocytes.

During the final stage of oocyte growth, the morphology of the oocyte nucleoli changes into a compact structure. The objective of this study was to determine the involvement of the proteasome, which is a large protein complex responsible for degrading intracellular proteins, in the nucleolar compaction. The mean nucleolar diameter of growing porcine oocytes (about 100 µm in diameter) was larger than that of fully grown (120 µm) oocytes (15.5 ± 0.3 vs. 13.2 ± 0.1 µm, P<0.05). When fully grown oocytes were treated with proteasome inhibitors, MG132 (10 and 20 µM) and lactacystin (100 and 200 µM), the nucleolar diameter significantly increased from 12.9 µm to 14.9-16.1 µm. In contrast, transcription inhibitors, actinomycin D (0.8-8 µM) and α-amanitin (10-100 µM) reduced the nucleolar diameter of growing oocytes to 9.4-12.4 µm. MG132 partially prevented this reduction in nucleolar diameter. These results suggest that the proteasome regulates the nucleolar size in porcine oocytes perhaps through the degradation of nucleolar proteins.

Involvement of mouse nucleoplasmin 2 in the decondensation of sperm chromatin after fertilization.

The tightly condensed chromatin of spermatozoa is rapidly decondensed after the spermatozoa enter oocytes. Although no factor involved in sperm chromatin decondensation (SCD) has been identified in mammals, it has been suggested that a factor related to SCD activity is present in the germinal vesicle (GV) of oocytes. Here, we found that the nucleolus-like body (NLB), which is a component of the GV, is involved in SCD in murine oocytes. When NLBs were microsurgically removed from GV-stage oocytes, SCD was significantly retarded in the paternal genome after fertilization following meiotic maturation. We found that the retardation of SCD in the NLB-removed oocytes was restored by the microinjection of mRNA encoding nucleoplasmin 2 (NPM2), a component of NLBs. Furthermore, SCD was retarded in the fertilized oocytes from Npm2-knockout females, and recombinant NPM2 alone could induce the SCD in vitro. These data provide evidence that NPM2 is involved in sperm chromatin remodeling in mammals.

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 oocyte's meiotic arrest, and that the full-grown oocyte nucleolus has lost the ability.

Loss of sister kinetochore co-orientation and peri-centromeric cohesin protection after meiosis I depends on cleavage of centromeric REC8.

Protection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required.

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 microm in diameter) and full-grown porcine oocytes (120 microm) 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.

IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

APC/CCdh1 Enables Removal of Shugoshin-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity.

In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata.

Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.

Reconstitution of female germ cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells and induced pluripotent stem cells in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, and cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ cell development in vitro.

Condensins I and II are essential for construction of bivalent chromosomes in mouse oocytes.

In many eukaryotes, condensins I and II associate with chromosomes in an ordered fashion during mitosis and play nonoverlapping functions in their assembly and segregation. Here we report for the first time the spatiotemporal dynamics and functions of the two condensin complexes during meiotic divisions in mouse oocytes. At the germinal vesicle stage (prophase I), condensin I is present in the cytoplasm, whereas condensin II is localized within the nucleus. After germinal vesicle breakdown, condensin II starts to associate with chromosomes and becomes concentrated onto chromatid axes of bivalent chromosomes by metaphase I. REC8 "glues" chromosome arms along their lengths. In striking contrast to condensin II, condensin I localizes primarily around centromeric regions at metaphase I and starts to associate stably with chromosome arms only after anaphase I. Antibody injection experiments show that condensin functions are required for many aspects of meiotic chromosome dynamics, including chromosome individualization, resolution, and segregation. We propose that the two condensin complexes play distinctive roles in constructing bivalent chromosomes: condensin II might play a primary role in resolving sister chromatid axes, whereas condensin I might contribute to monopolar attachment of sister kinetochores, possibly by assembling a unique centromeric structure underneath.

The nucleolus in the mouse oocyte is required for the early step of both female and male pronucleus organization.

During oocyte growth in the ovary, the nucleolus is mainly responsible for ribosome biogenesis. However, in the fully-grown oocyte, all transcription ceases, including ribosomal RNA synthesis, and the nucleolus adopts a specific monotonous fibrillar morphology without chromatin. The function of this inactive nucleolus in oocytes and embryos is still unknown. We previously reported that the embryo lacking an inactive nucleolus failed to develop past the first few cleavages, indicating the requirement of a nucleolus for preimplantation development. Here, we reinjected the nucleolus into oocytes and zygotes without nucleoli at various time points to examine the timing of the nucleolus requirement during meiosis and early embryonic development. When we put the nucleolus back into oocytes lacking a nucleolus at the germinal vesicle (GV) stage and at second metaphase (MII), these oocytes were fertilized, formed pronuclei with nucleoli and developed to full term. When the nucleolus was reinjected at the pronucleus (PN) stage, most of the reconstructed zygotes cleaved and formed nuclei with nucleoli at the 2-cell stage, but the rate of blastocyst formation and the numbers of surviving pups were profoundly reduced. Moreover, the zygotes without nucleoli showed a disorder of higher chromatin organization not only in the female pronucleus but also, interestingly, in the male pronucleus. Thus, the critical time point when the nucleolus is required for progression of early embryonic development appears to be at the point of the early step of pronucleus organization.

The maternal nucleolus is essential for early embryonic development in mammals.

With fertilization, the paternal and maternal contributions to the zygote are not equal. The oocyte and spermatozoon are equipped with complementary arsenals of cellular structures and molecules necessary for the creation of a developmentally competent embryo. We show that the nucleolus is exclusively of maternal origin. The maternal nucleolus is not necessary for oocyte maturation; however, it is necessary for the formation of pronuclear nucleoli after fertilization or parthenogenetic activation and is essential for further embryonic development. In addition, the nucleolus in the embryo produced by somatic cell nuclear transfer originates from the oocyte, demonstrating that the maternal nucleolus supports successful embryonic development.

Loss of Rec8 from chromosome arm and centromere region is required for homologous chromosome separation and sister chromatid separation, respectively, in mammalian meiosis.

Chromosome separation in meiosis I is different from those in mitosis and meiosis II in that homologs separate from each other in the former while sisters do so in the latter. We show here that meiosis-specific cohesin subunit Rec8 in mouse oocytes shows essentially the same pattern of localization to those reported in yeasts and mammalian spermatocytes; Rec8 along chromosome arm (armRec8) is lost at the metaphase I-to-anaphase I transition, although centromeric Rec8 (cenRec8) is maintained until the onset of anaphase II. Suppression of the loss of armRec8 by microinjection of anti-Rec8 antibody into the oocytes inhibits homolog separation but not the first polar body emission (cytokinesis). Similarly, the injection of anti-Rec8 antibody into metaphase II oocytes prevents sister separation in anaphase II after oocyte activation. These data demonstrate that the loss of armRec8 and cenRec8 is required for separation of homologs and sisters, respectively, but both are not required for other late mitotic events such as spindle elongation and cytokinesis in mouse oocytes. Further, by using some inhibitors for spindle assembly, proteasome and Topoisomerase II and overexpression of Securin, we propose that loss of armRec8 (homolog separation) and cytokinesis are suppressed until anaphase I by Securin whose destruction is regulated by spindle checkpoint-proteasome pathway, and that Topoisomerase II is required for homolog separation independently from such pathway.

Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDK1 and MAP kinase during maturation and fertilization of pig oocytes.

In amphibian oocytes, it is known that germinal vesicle (GV) materials are essential for sperm head decondensation but not for activation of MPF (CDK1 and cyclin B). However, in large animals, the role of GV materials in maturation and fertilization is not defined. In this study, we prepared enucleated pig oocytes at the GV stage and cultured them to examine the activation and inactivation of CDK1 and MAP kinase during maturation and after electro-activation. Moreover, enucleated GV-oocytes after maturation culture were inseminated or injected intracytoplasmically with spermatozoa to examine their ability to decondense the sperm chromatin. Enucleated oocytes showed similar activation/inactivation patterns of CDK1 and MAP kinase as sham-operated oocytes during maturation and after electro-stimulation or intracytoplasmic sperm injection. During the time corresponding to MI/MII transition of sham-operated oocytes, enucleated oocytes inactivated CDK1. However, penetrating sperm heads in enucleated oocytes did not decondense enough to form male pronuclei. To determine whether the factor(s) involved in sperm head decondensation remains associated with the chromatin after GV breakdown (GVBD), we did enucleation soon after GVBD (corresponding to pro-metaphase I, pMI) to remove only chromosomes. The injected sperm heads in pMI-enucleated oocytes decondensed and formed the male pronuclei. These results suggest that in pig oocytes, GV materials are not required for activation/inactivation of CDK1 and MAP kinase, but they are essential for male pronucleus formation.

Effect of caffeine on meiotic maturation of porcine oocytes.

This study was conducted to evaluate the effect of caffeine on the meiotic maturation of porcine oocytes. Oocyte-cumulus complexes were collected from slaughterhouse-derived ovaries and cultured for 24, 32 or 48 h in medium 199 supplemented with 10% fetal calf serum, 10 microg/ml FSH, 50 microg/ml sodium pyruvate and 50 microg/ml gentamicin in the presence or absence of 2.5 mM caffeine. Caffeine inhibited the meiotic resumption of pig oocytes effectively after 24 h of culture, and 95.5% of oocytes were arrested at the germinal vesicle (GV) stage (control 17.8%, p < 0.05). Prolonged culture with caffeine up to 32 h or 48 h, however, resulted in a significant decrease in the inhibitory effect (GV: 13.8% and 8.2%). The number of oocytes at metaphase II after 48 h of culture in the presence of caffeine was significantly lower than that in the control medium (65.3% vs 94.7%, p < 0.05). The withdrawal of caffeine after 24 h of culture resulted in the resumption of meiotic maturation, and the oocytes reached metaphase II after 48 h. However, the ability of caffeine-treated oocytes to develop to blastocysts after artificial activation was lower than that of the control (5.5% vs 9.1%, p < 0.05). Caffeine treatment significantly increased cAMP levels in the oocytes after 24 h of culture, while both Cdc2 kinase and MAP kinase activation were inhibited in the oocytes. These results suggest that caffeine, similarly to other purine derivatives, prolongs the meiotic arrest of porcine oocytes at the GV stage, perhaps by its action of increasing the cAMP level and by the suppression of Cdc2 kinase and MAP kinase activities in the oocytes.