Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.

Biallelic variant in cyclin B3 is associated with failure of maternal meiosis II and recurrent digynic triploidy

BackgroundTriploidy is one of the most common chromosome abnormalities affecting human gestation and accounts for an important fraction of first-trimester miscarriages. Triploidy has been demonstrated in a few cases of recurrent pregnancy loss (RPL) but its molecular mechanisms are unknown. This study aims to identify the genetic cause of RPL associated with fetus triploidy.MethodsWe investigated genomic imprinting, genotyped sequence-tagged site (STS) markers and performed exome sequencing in a family including two sisters with RPL. Moreover, we evaluated oocyte maturation in vivo and in vitro and effect of the candidate protein variant in silico.ResultsWhile features of hydatidiform mole were excluded, the presence of triploidy of maternal origin was demonstrated in the fetuses. Oocyte maturation was deficient and all the maternally inherited pericentromeric STS alleles were homozygous in the fetuses. A deleterious missense variant (p.V1251D) of the cyclin B3 gene (CCNB3) affecting a residue conserved in placental mammals and located in a region that can interact with the cyclin-dependent kinase 1 or cyclin-dependent kinase 2 cosegregated in homozygosity with RPL.ConclusionHere, we report a family in which a damaging variant in cyclin B3 is associated with the failure of oocyte meiosis II and recurrent fetus triploidy, implicating a rationale for CCNB3 testing in RPL.

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

ABSTRACTIn mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its Cdc20 co-activator. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 is required for meiotic anaphase in flies, worms and vertebrates, but whether it activates the APC/C is unclear. We found that Drosophila Cyclin B3 (CycB3) collaborates with PP2A-B55/Tws in embryonic development, indicating that CycB3 also promotes anaphase in mitosis. Moreover, CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its co-activators. We propose that CycB3-Cdk1 directly phosphorylates the APC/C to activate it in both meiosis and mitosis.

Characterization and localization of cyclin B3 transcript in both oocyte and spermatocyte of the rainbow trout (Oncorhynchus mykiss)

B-type cyclins are regulatory subunits with distinct roles in the cell cycle. To date, at least three subtypes of B-type cyclins (B1, B2, and B3) have been identified in vertebrates. Previously, we reported the characterization and expression profiles of cyclin B1 and B2 during gametogenesis in the rainbow trout (Oncorhynchus mykiss). In this paper, we isolated another subtype of cyclin B, cyclin B3 (CB3), from a cDNA library of the rainbow trout oocyte. The full-length CB3 cDNA (2,093 bp) has an open reading frame (1,248 bp) that encodes a protein of 416 amino acid residues. The CB3 transcript was widely distributed in all the examined tissues, namely, eye, gill, spleen, brain, heart, kidney, stomach, skin, muscle, and, especially, gonad. Northern blot analysis indicated only one form of the CB3 transcript in the testis and ovary. In situ hybridization revealed that, in contrast to cyclin B1 and B2 transcripts, CB3 transcripts were localized in the oocytes, spermatocytes, and spermatogonia. These findings strongly suggest that CB3 plays a role not only as a mitotic cyclin in spermatogonial proliferation during early spermatogenesis but also during meiotic maturation of the spermatocyte and oocyte in the rainbow trout.

Cyclin B3 is dispensable for mouse spermatogenesis

Cyclins, as regulatory partners of cyclin-dependent kinases (CDKs), control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Compared to mitosis, relatively little is known about how cyclin-CDK complexes control meiosis, the specialized cell division that generates gametes for sexual production. Mouse cyclin B3 was previously shown to have expression restricted to the beginning of meiosis, making it a candidate to regulate meiotic events. Indeed, female mice lacking cyclin B3 are sterile because oocytes arrest at the metaphase-to-anaphase transition of meiosis I. However, whether cyclin B3 functions during spermatogenesis was untested. Here, we found that males lacking cyclin B3 are fertile and show no detectable defects in spermatogenesis based on histological analysis of seminiferous tubules. Cytological analysis further showed no detectable defects in homologous chromosome synapsis or meiotic progression, and suggested that recombination is initiated and completed efficiently. Moreover, absence of cyclin B3 did not exacerbate previously described meiotic defects in mutants deficient for cyclin E2, suggesting a lack of redundancy between these cyclins. Thus, unlike in females, cyclin B3 is not essential for meiosis in males despite its prominent meiosis-specific expression.

Cyclin B3 is required for metaphase to anaphase transition in oocyte meiosis I

Meiosis with a single round of DNA replication and two successive rounds of chromosome segregation requires specific cyclins associated with cyclin-dependent kinases (CDKs) to ensure its fidelity. But how cyclins control the distinctive meiosis is still largely unknown. In this study, we explored the role of cyclin B3 in female meiosis by generating Ccnb3 mutant mice via CRISPR/Cas9. Ccnb3 mutant oocytes characteristically arrested at metaphase I (MetI) with normal spindle assembly and lacked enough anaphase-promoting complex/cyclosome (APC/C) activity, which is spindle assembly checkpoint (SAC) independent, to initiate anaphase I (AnaI). Securin siRNA or CDK1 inhibitor supplements rescued the MetI arrest. Furthermore, CCNB3 directly interacts with CDK1 to exert kinase function. Besides, the MetI arrest oocytes had normal development after intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA), along with releasing the sister chromatids, which implies that Ccnb3 exclusively functioned in meiosis I, rather than meiosis II. Our study sheds light on the specific cell cycle control of cyclins in meiosis.

Cyclin B3 promotes anaphase I onset in oocyte meiosis

Meiosis poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we show that female mice genetically ablated for cyclin B3 are viable—indicating that the protein is dispensable for mitotic divisions—but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective anaphase-promoting complex/cyclosome (APC/C) activation, including no separase activity, high CDK1 activity, and high cyclin B1 and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3–associated kinase activity. Cyclin B3 homologues from frog, zebrafish, and fruit fly rescue meiotic progression in cyclin B3–deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.

Cyclin B3 promotes APC/C activation and anaphase I onset in oocyte meiosis

As obligate kinase partners, cyclins control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Meiosis, the cell division that generates gametes for sexual reproduction, poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous, temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. Here, we delineate an essential function for mouse cyclin B3 in the first meiotic division of oocytes. Females genetically ablated for cyclin B3 are viable, indicating the protein is dispensable for mitotic divisions, but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective APC/C activation, including no separase activity and high MPF, cyclin B1, and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded and arrest can be suppressed by inhibiting MPF kinase. Cyclin B3 is itself targeted for degradation by the APC/C. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Collectively, our findings indicate that cyclin B3 is essential for oocyte meiosis because it fine-tunes APC/C activity as a kinase-activating CDK partner. Cyclin B3 homologs from frog, zebrafish, and fruitfly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin.