Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II

Abstract

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high-resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.

Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition

“…This reduced Plk1 activity leads to a corresponding reduction in pericentromeric Bub1 and Sgo2, allowing for Rec8 phosphorylation and cleavage at anaphase II. This model is consistent with recent findings that separase activity at meiosis I is required for the deprotection of pericentromeric cohesin and individualization of sister kinetochores during anaphase I (Gryaznova et al, 2021;Ogushi et al, 2020). Finally, as germ cells exit meiosis, Meikin is completely eliminated through APC/C-mediated degradation (Figure 6A), returning kinetochore regulation to a mitotic state.…”

Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes

“…Whereas control oocytes harbored only paired sister chromatids (dyads), the majority of oocytes treated with Aurora B/C inhibitors contained several bivalents (paired chromosomes), indicating failures in meiosis I chromosome segregation (Figure 1C-G). This phenotype is not due to absence of error correction resulting in chromosomes being attached to only one pole, because -as we have shown previously-bivalents attached to a monopolar spindle still separate homologous chromosomes 20 . Hence, Aurora B/C kinase may be involved in efficient arm cohesin removal 4 in meiosis I.…”

Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes

“…Whereas control oocytes harbored only paired sister chromatids (dyads), the majority of oocytes treated with Aurora B/C inhibitors contained several bivalents (paired chromosomes), indicating failures in meiosis I chromosome segregation (Figure 1c-g). This phenotype is not due to absence of error correction resulting in chromosomes being attached to only one pole, because -as we have shown previously-bivalents attached to a monopolar spindle still separate homologous chromosomes [20]. Hence, Aurora B/C kinase may be involved in efficient arm cohesin removal in meiosis I.…”

“…But before addressing this issue with phospho-specific antibodies, we wanted to determine the amount of endogenous centromeric Rec8 present in meiosis I and maintained until meiosis II, to check whether we could actually expect to distinguish distinctly phosphorylated fractions of endogenous Rec8 around the centromere, by immunostaining. We have discovered recently that an event we termed kinetochore individualization takes place in anaphase I and may require cleavage of centromeric Rec8 [20]. Indeed, additionally to armand pericentromere-localized Rec8, a third fraction at the centromere was recently shown to be removed at exit from meiosis I [32], and may therefore also be modified by phosphorylation already in metaphase I.…”

“…Indeed, an unspecific signal colocalizing with CREST was detected on mitotic and meiotic spreads with serum C purified against both phosphorylation sites, whereas in addition to this unspecific signal, another signal in-between sister centromere dots exactly where centromeric cohesin is localized (Figure 4a), was detected in meiosis II (Figure S3d). Hence we could not assess pRec8 staining in the centromere region in meiosis I when sister kinetochores are fused [20] with this antibody because we could not distinguish the specific from the unspecific signal at that location. Together with the fact that 80 % of centromeric Rec8 are removed in meiosis I already (Figure 4a) we thus decided to limit our analysis to arm cohesin, as the pattern of cross-shaped staining is only observed for proteins of the cohesin complex.…”