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

APC/C(Cdh1) 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/C(Cdc20)) 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/C(Cdh1)) 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/C(Cdh1) 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

Maturation and fertilization of pig enucleolated oocytes

ABSTRACT- Fully grown oocytes have a large nucleus, the germinal vesicle (GV), which includes a single large nucleolus. Since the nucleolus is transcriptionally inactive and disappears during oocyte maturation, the role of the nucleolus in oocyte maturation and subsequent fertilization has not been elucidated. In the present study, pig oocytes at the GV-stage were enucleolated (removed the nucleolus by micromanipulation), and subjected to intracytoplasmic sperm injection (ICSI). Enucleolated oocytes underwent germinal vesicle breakdown after 12 hr of culture (21/35). After 30 hr, they matured to metaphase II (MII) with a normal spindle (23/45). Sham-operated oocytes underwent germinal vesicle breakdown after 12 hr (22/35), and matured to MII (28/39) in the similar time course to that of enucleolated oocytes. Activities of Cdc2 kinase and MAP kinase were increased after 12 hr of maturation culture and the high activities were maintained until 30 hr in both enucleolated and sham-operated oocytes. When the matured oocytes were electro-stimulated, Cdc2 kinase activity in both group oocytes decreased to the basal level after 8 hr, and the activity of MAP kinase decreased after Cdc2 kinase inactivation. Enucleolated and matured oocytes were subjected to ICSI. Sham-operated oocytes formed male and female pronuclei (33/73) having the nucleoli (29/33) 24 hr after sperm injection, and the enucleolated oocytes form the male and female procnuclei (36/76) 24 hr after ICSI but no nucleolus was observed in both pronuclei (0/36). Enucleolated oocytes and nucleoli at GV-stage were cultured separately for 26 hr. Enucleolated oocytes matured to MII, while nucleoli were still intact. The cultured nucleoli were put back into enucleolated MII oocytes after 26 hr culture. These oocytes were cultured further 4 hr and subjected to ICSI. The enucleolated MII oocytes returned the nucleoli formed the male and female pronuclei with nucleoli 24 hr after ICSI. In summary, the prominent nucleoli in pig GV oocytes are not required for oocyte maturation and fertilization, and activation and inactivation of Cdc2 kinase and MAP kinase. However, it is indispensable for nucleolus formation in male and female pronuclei. This suggests that nucleoli in pronuclei of fertilized oocytes are maternal origin.[...

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

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