Function of cyclins in regulating the mitotic and meiotic cell cycles in male germ cells

The unique cell cycles that characterize various aspects of the differentiation of germ cells provide a unique opportunity to understand heretofore elusive aspects of the in vivo function of cell cycle regulators. Key components of the cell cycle machinery are the regulatory sub-units, the cyclins, and their catalytic partners, the cyclin-dependent kinases. Some of the cyclins exhibit unique patterns of expression of germ cells that suggest possible concomitant distinct functions, predictions that are being explored by targeted mutagenesis in mouse models. A novel, meiosis-specific function has been shown for one of the A-type cyclins, cyclin A1. Embryonic lethality has obviated understanding of the germline functions of cyclin A2 and cyclin B1, while yet other cyclins, although expressed at specific stages of germ cell development, may have less essential function in the male germline.     

A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms

Populations of hormone-stimulated starfish oocytes and fertilized sea urchin eggs undergo synchronous meiotic and mitotic divisions. We have studied the requirement for protein phosphorylation during these events by testing the effects of 6-dimethylaminopurine (6-DMAP) upon the incorporation of [32P]orthophosphate. It was found that 6-DMAP blocked meiosis reinitiation and early cleavage and simultaneously inhibited protein phosphorylation, without changing the rate of [35S]methionine incorporation or pattern of protein synthesis. The protein, cyclin (54 kDa in starfish and 57 kDa in sea urchin), continues to be synthesized in the presence of 6-DMAP. This protein is destroyed at first and second cell cycles when 6-DMAP is added 30 min following fertilization but not when this drug is present before fertilization. Thus, cyclin breakdown does not depend on the completion of the nuclear events of M-phase, and its time of breakdown is set at an early step between fertilization and first cleavage. Using tubulin immunostaining, we found that 6-DMAP did not affect the cortical microtubules and resting female centrioles of prophase-arrested starfish oocytes, whereas it induced a precocious disappearance of spindle fibers when applied to hormone-stimulated oocytes. While an early addition of 6-DMAP precluded nuclear breakdown and spindle formation in both systems, a late treatment always allowed chromosome separation and centriole separation. Under these conditions pericentriolar tubulin persisted and could organize new spindles after the inhibitor was removed. It is suggested that (1) the assembly of cortical and centriolar-associated microtubules is not controlled by the same factors as spindle-associated tubulin; (2) specific proteins which are required for the cell to enter the following M-phase can become operative only via a process depending upon protein phosphorylation; (3) microtubule-associated kinases may play an important role in MPF function and spindle dynamics.     

The roles of Drosophila cyclins A and B in mitotic control

We have cloned, sequenced, and characterized the expression of a Drosophila cyclin B gene. The independent evolutionary conservation of A- and B-type cyclins implies that they have distinct roles. Indeed, in mutant embryos deficient in cyclin A, cells that accumulate only cyclin B do not enter mitosis. Thus, in vivo, cyclin B is not sufficient for mitosis. Furthermore, we find that the two cyclins are coexpressed in all proliferating cells throughout development. Though lacking a formal demonstration that cyclin B is essential as it is in other organisms, we propose that each of these proteins fulfills a distinct and essential role in the cell cycle.     

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.     

Characterization of protein kinases in mitotic and meiotic cell extracts

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.     

Protein kinase cascades in meiotic and mitotic cell cycle control

Eukaryotic cell cycle progression during meiosis and mitosis is extensively regulated by reversible protein phosphorylation. Many cell surface receptors for mitogens are ligand-stimulated protein-tyrosine kinases that control the activation of a network of cytoplasmic and nuclear protein-serine (threonine) kinases. Over 30 plasma membrane associated protein-tyrosine kinases are encoded by proto-oncogenes, i.e., genes that have the potential to facilitate cancer when disregulated. Proteins such as ribosomal protein S6, microtubule-associated protein-2, myelin basic protein, and casein have been used to detect intracellular protein-serine (threonine) kinases that are activated further downstream in growth factor signalling transduction cascades. Genetic analysis of yeast cell division control (cdc) mutants has revealed another 20 or so protein-serine (threonine) kinases. One of these, specified by the cdc-2 gene in Schizosaccharomyces pombe, has homologs that are stimulated during M phase in maturing sea star and frog oocytes and mammalian somatic cells. Furthermore, during meiotic maturation in these echinoderm and amphibian oocytes, this is followed by activation of many of the same protein-serine (threonine) kinases that are stimulated when quiescent mammalian somatic cells are prompted with mitogens to traverse from G0 to G1 phase. These findings imply that a similar protein kinase cascade may oversee progression at multiple points in the cell cycle.     

The degradation of two mitotic cyclins contributes to the timing of cytokinesis

BACKGROUND: Cytokinesis occurs just as chromosomes complete segregation and reform nuclei. It has been proposed that cyclin/Cdk kinase inhibits cytokinesis until exit from mitosis; however, the timer of cytokinesis has not been experimentally defined. Whereas expression of a stable version of Drosophila cyclin B blocks cytokinesis along with numerous events of mitotic exit, stable cyclin B3 allows cytokinesis even though it blocks late events of mitotic exit. We examined the interface between mitotic cyclin destruction and the timing of cytokinesis. RESULTS: In embryonic mitosis 14, the cytokinesis furrow appeared 60 s after the metaphase/anaphase transition and closed 90 s later during telophase. In cyclin B or cyclin B3 mutant cells, the cytokinesis furrow appeared at an earlier stage of mitosis. Expression of stable cyclin B3 delayed and prolonged furrow invagination; nonetheless, cytokinesis completed during the extended mitosis. Reduced function of Pebble, a Rho GEF required for cytokinesis, also delayed and slowed furrow invagination, but incomplete furrows were aborted at the time of mitotic exit. In functional and genetic tests, cyclin B and cyclin B3 inhibited Pebble contributions to cytokinesis. CONCLUSIONS: Temporal coordination of mitotic events involves inhibition of cytokinesis by cyclin B and cyclin B3 and punctual relief of the inhibition by destruction of these cyclins. Both cyclins inhibit Pebble-dependent activation of cytokinesis, whereas cyclin B can inhibit cytokinesis by additional modes. Stable cyclin B3 also blocks the later return to interphase that otherwise appears to impose a deadline for the completion of cytokinesis.     

Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.     

The anaphase-promoting complex/cyclosome inhibitor Emi2 is essential for meiotic but not mitotic cell cycles

Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown. Here we show that during oocyte maturation, Emi2 appears only after metaphase I, and its level peaks at CSF arrest (metaphase II). In M phase, Emi2 undergoes a phosphorylation-dependent electrophoretic shift. Microinjection of antisense oligonucleotides against Emi2 into stage VI oocytes blocks progression through meiosis II and the establishment of CSF arrest. Recombinant Emi2 rescues CSF arrest in these oocytes and also causes CSF arrest in egg extracts and in blastomeres of two-cell embryos. Fertilization triggers rapid, complete degradation of Emi2, but it is resynthesized in the first embryonic cell cycle to reach levels 5-fold lower than during CSF arrest. However, depletion of the protein from cycling egg extracts does not prevent mitotic cell cycle progression. Thus, Emi2 plays an essential role in meiotic but not mitotic cell cycles.     

Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos.

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.     

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin''s spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo''s negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.     

Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.

Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.     

The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis

BACKGROUND: Degradation of the mitotic cyclins is a hallmark of the exit from mitosis. Induction of stable versions of each of the three mitotic cyclins of Drosophila, cyclins A, B, and B3, arrests mitosis with different phenotypes. We tested a recent proposal that the destruction of the different cyclins guides progress through mitosis. RESULTS: Real-time imaging revealed that arrest phenotypes differ because each stable cyclin affects specific mitotic events differently. Stable cyclin A prolonged or blocked chromosome disjunction, leading to metaphase arrest. Stable cyclin B allowed the transition to anaphase, but anaphase A chromosome movements were slowed, anaphase B spindle elongation did not occur, and the monooriented disjoined chromosomes began to oscillate between the spindle poles. Stable cyclin B3 prevented normal spindle maturation and blocked major mitotic exit events such as chromosome decondensation but nonetheless allowed chromosome disjunction, anaphase B, and formation of a cytokinetic furrow, which split the spindle. CONCLUSIONS: We conclude that degradation of distinct mitotic cyclins is required to transit specific steps of mitosis: cyclin A degradation facilitates chromosome disjunction, cyclin B destruction is required for anaphase B and cytokinesis and for directional stability of univalent chromosome movements, and cyclin B3 degradation is required for proper spindle reorganization and restoration of the interphase nucleus. We suggest that the schedule of degradation of cyclin A, cyclin B, and then cyclin B3 contributes to the temporal coordination of mitotic events.     

Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte

The pattern of protein synthesis in oocytes of starfish Marthasterias glacialis changes during 1-methyladenine-induced meiotic maturation. One of the newly synthesized proteins, a major 54-kDa polypeptide, was synthesized continuously after activation but was destroyed abruptly just before appearance of the polar bodies at each meiotic division. This protein thus resembles the cyclin proteins identified in cleaving sea urchin and clam embryos. RNA extracted from oocytes before and after maturation encoded virtually identical polypeptides when translated in the reticulocyte lysate. However, there was poor correspondence between the in vitro translation products and the labelling pattern of intact cells. There was no exact in vitro counterpart to the in vivo-labelled cyclin. Instead, a major polypeptide of 52 kDa was seen which appears to be a precursor of the 54-kDa form of cyclin. The 52-kDa polypeptide was identified as cyclin by hybrid arrest of translation. Cyclin mRNA is ot translated to a significant extent before oocyte activation and is present in oocytes as nonadenylated form. It becomes polyadenylated when the oocytes mature. This behavior is also seen in the case of the mRNA for the small subunit of ribonucleotide reductase, another abundant maternal mRNA whose translation is activated at maturation.     

Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3.

While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition.     

The Drosophila kinesin-like protein KLP67A is essential for mitotic and male meiotic spindle assembly

We have performed a mutational analysis together with RNA interference to determine the role of the kinesin-like protein KLP67A in Drosophila cell division. During both mitosis and male meiosis, Klp67A mutations cause an increase in MT length and disrupt discrete aspects of spindle assembly, as well as cytokinesis. Mutant cells exhibit greatly enlarged metaphase spindle as a result of excessive MT polymerization. The analysis of both living and fixed cells also shows perturbations in centrosome separation, chromosome segregation, and central spindle assembly. These data demonstrate that the MT plus end-directed motor KLP67A is essential for spindle assembly during mitosis and male meiosis and suggest that the regulation of MT plus-end polymerization is a key determinant of spindle architecture throughout cell division.     

A novel destruction sequence targets the meiotic regulator Spo13 for anaphase-promoting complex-dependent degradation in anaphase I

The anaphase-promoting complex (APC) or cyclosome is a multisubunit ubiquitin-protein ligase that ubiquitinates and thereby promotes the destruction of the mitotic cyclins and the separase inhibitor, securin. The contributions of the APC to progression through the meiotic program are not clear. To clarify the function of the APC in meiosis, we screened several yeast meiotic proteins as APC substrates in vitro. We found that the meiotic regulator Spo13 is an APC substrate that is degraded during anaphase I. Spo13 is expressed only in meiotic cells, where it has multiple functions, including the promotion of monopolar chromosome attachment in the first division. Spo13 ubiquitination by the APC depends on an LxExxxN sequence (residues 26-32) that is distinct from previously described destruction sequences of APC substrates. Mutation of one residue, leucine 26, prevented Spo13 ubiquitination by the APC in vitro and stabilized the protein through the meiotic divisions. Analysis of meiotic progression and spore viability of yeast containing the stabilized Spo13 mutant revealed no significant defects, indicating that Spo13 destruction in anaphase I is not essential for meiosis. We propose that Spo13 destruction is one of multiple mechanisms underlying the switch from monopolar to bipolar chromosome attachment between the meiotic divisions.