Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase-anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.
The meiosis-specific kleisin cohesin subunit, RAD21L, may participate in synapsis initiation and crossover recombination between homologous chromosomes.
In addition to inter-chromatid cohesion, mitotic and meiotic chromatids must have three physical properties: compaction into 'threads' roughly co-linear with their DNA sequence, intra-chromatid cohesion determining their rigidity, and a mechanism to promote sister chromatid disentanglement. A fundamental issue in chromosome biology is whether a single molecular process accounts for all three features. There is universal agreement that a pair of Smc-kleisin complexes called condensin I and II facilitate sister chromatid disentanglement, but whether they also confer thread formation or longitudinal rigidity is either controversial or has never been directly addressed respectively. We show here that condensin II (beta-kleisin) has an essential role in all three processes during meiosis I in mouse oocytes and that its function overlaps with that of condensin I (gamma-kleisin), which is otherwise redundant. Pre-assembled meiotic bivalents unravel when condensin is inactivated by TEV cleavage, proving that it actually holds chromatin fibres together.There is no more fundamental or indeed marked transformation of chromatin fibres than that which precedes and facilitates their segregation during mitosis or meiosis. From occupying a broad and amorphous territory within the interphase nucleus1, individual DNA molecules metamorphose into thread-like sister chromatids2. Importantly, these chromatids possess sufficient plasticity along their longitudinal axes to prevent breakage of entangled sisters and yet enough rigidity to ensure that the chromatid is not unravelled when kinetochores are pulled towards the poles by microtubules. Remarkably, the segregation of most sister DNA sequences from each other (pre-segregation) takes place in the complete absence of spindle forces, facilitating sister chromatid disjunction at the onset of anaphase3. The metamorphosis of interphase chromatin into pre-segregated chromatids is known as chromosome condensation. However, this term does not adequately convey the reality that Competing Financial InterestsThe authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nat Cell Biol. Author manuscript; available in PMC 2017 January 03. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts chromosome compaction generates threads not balls of chromatin. It also neglects the actuality that thread formation is accompanied both by pre-segregation and the generation of longitudinal rigidity4.Cohesion holding sister DNAs together is mediated by a ring-shaped complex called cohesin, formed by the binding of the amino-and carboxy-terminal domains of an alphakleisin subunit5 to the ATPase heads at the vertices of V-shaped SMC1/SMC3 heterodimers. Pioneering work using Xenopus extracts showed that the metamorphosis of sperm chromatin into chromatids depends on condensins, cohesin-like ring complexes formed by the interconnection of ATPases at the vertices of SMC2/SMC4 heterodimers by either beta-(condensin II) or gamma-(condensin I) kleisin...
This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.
In mouse oocytes, condensin I localizes around centromeric regions, whereas condensin II is concentrated onto chromatid axes of Meta-I bivalent chromosomes. Both condensins are required for many aspects of meiotic chromosome dynamics, including individualization, resolution, and segregation, as well as monopolar attachment of sister kinetochores.
Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylatred around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.
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.
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