Pyruvate prevents aging of mouse oocytes.

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus-oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.

Fertilization differently affects the levels of cyclin B1 and M-phase promoting factor activity in maturing and metaphase II mouse oocytes.

Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing and metaphase II mouse oocytes in two distinct ways. In metaphase II oocytes, it leads to a Ca(2)(+)-dependent, continuous degradation of cyclin B1 and inactivation of cyclin dependent kinase (CDC2A)-cyclin B1 complex (MPF). In this paper, we show that neither mono- nor polyspermic fertilization of prometaphase I and metaphase I oocytes triggered degradation of cyclin B1. However, polyspermic fertilization of prometaphase I oocytes led to a transient decrease in MPF activity that lasted for 2 h. The inactivation of MPF in polyspermic prometaphase I oocytes did not depend on the fertilization-induced increase in the cytoplasmic concentration of free Ca(2)(+) ions, but was caused, at least in part, by dephosphorylation of CDC2A at threonine 161 (Thr161). We found that polyspermic fertilization did not affect glutathione levels in prometaphase I oocytes, and concluded that the decrease in MPF activity and dephosphorylation of CDC2A at Thr161 in polyspermic prometaphase I oocytes were not caused by a change in the redox status of the cell induced by an introduction of excessive amount of sperm protamines. Instead, we propose that inactivation of MPF activity in polyspermic maturing oocytes is caused by a change in nucleo-cytoplasmic ratio that leads to a 'titration' of kinases and phosphatases responsible for keeping MPF in an active state. This idea is supported by the finding that oocytes fused with thymocytes rather than spermatozoa also showed a transient decrease in MPF activity.

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells.

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.

The effect of three-dimensional demineralized bone matrix on in vitro cumulus-free oocyte maturation.

The physiological role of cumulus cell surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. Collagen-based demineralized bone matrix (DBM) is a valuable biomaterial for the three-dimensional (3-D) cell culture. The present study was designed to determine whether in vitro maturation (IVM) of cumulus-free oocytes in mice could be improved by using the 3-D DBM co-culture system. The results indicated that the denuded oocytes cultured in 3-D DBM co-culture system with cumulus cells showed close similarity of cortical granules (CGs) distribution pattern, had more normal maturation-promoting factor (MPF) level and zona pellucida (ZP) hardening level to the in vivo matured oocytes, and the best preimplantation development after being activated by in vitro fertilization (IVF) or parthenogenetic activation. Thus, 3-D DBM collagen scaffold could serve as a tool for fundamental in vitro studies of cells or tissues under the environment that closely assembles the in vivo conditions.

Demecolcine-assisted enucleation for bovine cloning.

The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning.

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes.

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli.

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

The dynamics of cyclin B1 distribution during meiosis I in mouse oocytes.

Cdk1-cyclin B1 kinase activity drives oocytes through meiotic maturation. It is regulated by the phosphorylation status of cdk1 and by its spatial organisation. Here we used a cyclin B1-green fluorescent protein (GFP) fusion protein to examine the dynamics of cdk1-cyclin B1 distribution during meiosis I (MI) in living mouse oocytes. Microinjection of cyclin B1-GFP accelerated germinal vesicle breakdown (GVBD) and, as previously described, overrides cAMP-mediated meiotic arrest. GVBD was pre-empted by a translocation of cyclin B1-GFP from the cytoplasm to the germinal vesicle (GV). After nuclear accumulation, cyclin B1-GFP localised to the chromatin. The localisation of cyclin B1-GFP is governed by nuclear import and export. In GV intact oocytes, cyclin export was demonstrated by showing that cyclin B1-GFP injected into the GV is exported to the cytoplasm while a similar size dextran is retained. Import was revealed by the finding that cyclin B1-GFP accumulated in the GV when export was inhibited using leptomycin B. These studies show that GVBD in mouse oocytes is sensitive to cyclin B1 abundance and that the changes in distribution of cyclin B1 contribute to progression through MI.

[The activity of M-phase promoting factor in oral normal tissue and tumor].

OBJECTIVE: To investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and the relationship between MPF and malignant degree. METHODS: The content and activity of MPF were assessed by immunobloting and Gollicano method. RESULTS: The cdc2 and cyclinB (two subunits of MPF) were found both in normal and tumor tissues, and their content in tumor was higher than normal tissues. Buccal carcinoma was 64% higher than normal tissues. The activity of MPF in carcinoma was higher than normal tissue and had positive relation with the malignant extent. CONCLUSIONS: The content and activity of MPF in tumor are higher than normal tissue. PKC can activate MPF. These results show PKC may promote tumor proliferation by activating MPF and also, the activity of MPF has some relation with malignant extent.

Tuning the cell cycle: a model based on averaging.

The ability of intercellular communication and the basement membrane to revert the phenotypic behaviour of malignant cells suggests that such cells can be tuned to behave more benignly. In addition, the large variation in cell doubling times observed in tumour cells poses the question of whether or not cell doubling times, and hence, patient survival, can be lengthened by therapeutic intervention. In both cases, the understanding may be enhanced by obtaining a parsimonious and tractable model of the cell cycle which behaves appropriately and suggests a philosophical framework for addressing these complex issues. We introduce a simple two-dimensional model based on averaging cyclin and maturation promotion factor over a fast oscillating subsystem that exhibits the basic features of cellular division, and discuss the ramifications of the model.

Effect of dietary supplementation with a mixture of Vitamins C and E on fertilization of tertiary butyl hydroperoxide-treated oocytes and parthenogenetic activation in the mouse.

The present study aims to analyze the effect of dietary supplementation with a mixture of Vitamins C and E on fertilization and later development of tertiary butyl hydroperoxide (tBH)-treated mouse oocytes and on parthenogenetic activation of freshly ovulated mouse oocytes. We fed hybrid mice a standard diet supplemented or not supplemented with Vitamins C and E from the first day of weaning until the age of 12 weeks. We noted no significant effect of diet on fertilization rate, percentage of total and hatching blastocysts, total number of cells, mitotic index and percentage of apoptotic nuclei at 120 h post-insemination of oocytes incubated for 15 min in the presence of 0, 1, 5 and 10 microM tBH. Furthermore, diet did not affect the percentage of activated oocytes after treatment with Ca2+ ionophore, acid Tyrode's solution or ethanol. The percentage of parthenogenetically activated oocytes that progressed to the pronuclear stage was significantly higher in the antioxidant group. Oocytes from antioxidant females exhibited a significantly lower mitogen-activated protein kinase (MAPK) activity than oocytes from control females. We detected no significant differences between groups in M-phase-promoting factor (MPF) activity. These results show that oral administration of antioxidants decreases MAPK activity and increases the probability of reaching the pronuclear stage after parthenogenetic activation.

Biochemical and developmental evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised.

Our previous studies have shown that oocytes collected from prepubertal calves lack developmental competence. The overall objective of this study was to assess causes by comparing biochemical and physiologic changes during in vitro maturation of oocytes collected from ovaries of adult cattle at slaughter and from superstimulated calves (<6 mo old) by either laporotomy or ultrasound-guided follicular aspiration. Activity and/or concentrations of maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and inositol 1,4,5-trisphosphate receptor (IP(3)R) were determined by measuring phosphorylation of histone H-1 kinase, phosphorylation of myelin basic protein, or Western blotting, respectively, and were compared between oocytes collected from calves and for those collected from cows. The activities of MPF and MAPK and the relative amount of IP(3)R were significantly lower in calf oocytes. The physiologic significance of these observations was determined by assessing the developmental potential of embryos derived by reciprocal transfer of metaphase II (M-II) chromosomes between cow and calf ooplasts and transfer of adult cumulus cells (G0/G1) into cow and calf ooplasts. Procedural controls consisted of transfer of M-II between adult oocytes and parthenogenic activation of adult and calf oocytes. Adult parthenogenically activated oocytes cleaved and developed to blastocysts at a higher rate than did similarly activated calf oocytes (42.1% vs. 3.4%, P < 0.05). Cleavage was also higher in reciprocal M-II transfer embryos containing adult ooplasm (46.2% vs. 12.0%, P < 0.05). Cleavage (66.7% vs. 21.9%, P < 0.05) and development to blastocyst (20.1% vs. 4.8%, P < 0.05) of nuclear transfer embryos reconstructed from adult cumulus cells was higher after transfer to adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation.

Inactivation of M-phase promoting factor at exit from first embryonic mitosis in the rat is independent of cyclin B1 degradation.

Exit from M-phase and completion of cell division requires inactivation of M-phase promoting factor (MPF), a heterodimer composed of the regulatory cyclin B1 and the catalytic p34cdc2 kinase. Inactivation of MPF is associated with cyclin B1 degradation that is brought about by the ubiquitin-proteasome pathway. Our study examined the role of the proteasome in the first mitosis of rat embryos and its participation in the regulation of cyclin B1 degradation and MPF inactivation. We show that in the early zygote the proteasome is evenly distributed in the ooplasm and the nucleus, whereas during mitosis it accumulates on the spindle apparatus. We further demonstrate that inhibition of proteasomal catalytic activity prevents 1-cell embryos from undergoing mitosis. This mitotic arrest is associated with the presence of relatively high amounts of cyclin B1, which unexpectedly does not result in elevated MPF activity. Our findings strongly imply that completion of the first embryonic division depends on proteasomal degradation and that cyclin B1 is included among its target proteins. They also provide the first evidence that MPF inactivation at this stage of development is not solely dependent upon cyclin B1 degradation and is insufficient to allow the formation of the 2-cell embryo.

Inhibition of protein tyrosine phosphatases blocks calcium-induced activation of metaphase II-arrested oocytes of Xenopus laevis.

We have studied the effect of a protein tyrosine phosphatases (PTP) inhibitor on calcium-induced activation of Xenopus laevis oocytes arrested at metaphase II. Ammonium molybdate microinjection blocked pronucleus formation following A23187 treatment while cortical granules still underwent exocytosis. Pronuclei still occurred in ammonium molybdate-injected oocytes following 6-DMAP addition. Changes that usually occurred following A23187 exposure were inhibited in the presence of ammonium molybdate in the oocyte: MAPK dephosphorylation, p34(cdc2) rephosphorylation and cyclin B2 and p39(mos) proteolysis. These results suggest that a PTP is involved in the activation of the ubiquitin-dependent degradation machinery.

Effect of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, on the first mitotic divisions of the fertilized sea urchin egg.

We have reported earlier that the polyphosphoinositide messenger system may control mitosis in sea urchin eggs. Besides phospholipase C activation and its second messengers, phosphatidylinositol (PI) 3-kinase has been proposed to affect a wide variety of cellular processes in other cellular systems. Therefore, we have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. Our data presented here suggest that PI 3-kinase is present in sea urchin eggs. We found that wortmannin, an inhibitor of PI 3-kinase, led to arrest of the cell cycle. Chromosome condensation, nuclear envelope breakdown, microtubular aster polymerization, protein and DNA synthesis were not affected when fertilization was performed in the presence of the drug. However, maturation-promoting factor (MPF) activation was inhibited and centrosome duplication was perturbed preventing the formation of a bipolar mitotic spindle in wortmannin treated eggs. We discuss how PI 3-kinase might be involved in the cascade of events leading to the first mitotic divisions of the fertilized sea urchin egg.

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of 6-dimethylaminopurine.

The methods used to achieve blastomere cell cycle synchronization in embryos used as nuclear donors during embryo reconstruction have been largely unsuccessful. The aim of this study was to determine the reliability of 6-dimethylaminopurine (6-DMAP), an inhibitor of maturation promoting factor, to half and to synchronize blastomere division in cleavage stage bovine embryos. A second goal was to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h after insemination were treated with several concentrations of 6-DMAP for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology, DNA synthesis, histone H1 and scored for blastocyst formation and for hatching rate. They were subsequently fixed and the number of nuclei counted. Complete arrest of cell division was observed at concentrations of 3 mmol 6-DMAP l-1 and above. At these concentrations, interphase nuclei in arrest were noticeably larger compared with interphase nuclei of eight-cell control embryos. Removal from 6-DMAP led to release from cleavage arrest and was followed by synchronized mitosis, histone H1 kinase deactivation and re-entry into interphase within 4-5 h. Twenty-nine per cent of interphase nuclei were synthesizing DNA at the end of the 12 h treatment as indicated by BrdU analysis. At 2 h after removal from 6-DMAP, an abrupt decrease to 9% BrdU-positive nuclei was observed followed by an increase to 39% by 6 h and a decrease to 28% at 10 h. The ability of treated embryos to reach the blastocyst stage in vitro and the number of cells per blastocyst were reduced. These results indicate that 6-DMAP can reversibly arrest and synchronize cleavage to the fifth cell cycle in eight-cell bovine embryos. Although a decrease was observed in the proportion of blastocysts obtained after treatment, it is concluded that 6-DMAP is a useful tool for synchronization studies requiring donor nuclei at metaphase before fusion to recipient oocyte.

Use of a radioimmunoassay which detects C21 steroids with a 5beta-reduced, 3alpha-hydroxylated configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

Two radioimmunoassays (RIAs) have been developed which detect C21 (pregnane) steroids with a 5beta-reduced, 3alpha-hydroxyl (5beta, 3alpha) configuration. One RIA only detects 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one and 3alpha, 17-dihydroxy-5beta-pregnan-20-one, whilst the other detects a range of 5beta,3alpha steroids, including 5beta-pregnane-3alpha,17,20 beta-triol, a major metabolite of 17, 20beta-dihydroxy-4-pregnen-3-one, the putative oocyte maturation-inducing steroid in plaice Pleuronectes platessa. The RIAs, in conjunction with reverse-phase high-performance liquid chromatography (HPLC), have identified and quantified the steroids in plasma and urine of reproductively mature females. Total levels of 5beta,3alpha metabolites which can be extracted with diethyl ether (i.e., free steroids) are relatively low (<10 ng/ml). However, total levels of 5beta,3alpha metabolites released by solvolysis (i.e. , sulphated steroids) are very high (up to 1000 ng/ml in plasma and 20 microg/ml in urine). On HPLC, these metabolites have been identified (in order of their abundance in plasma) as: 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one, 5beta-pregnane-3alpha,17, 20beta-triol, 5beta-pregnane-3alpha,17,20alpha-triol, 3alpha,11beta, 17,21-tetrahydroxy-5beta-pregnane-20-one, and 3alpha, 17-dihydroxy-5beta-pregnan-20-one. Levels of the first three steroids are significantly elevated in female plaice undergoing natural or gonadotrophin-induced final oocyte maturation.

Use of a radioimmunoassay which detects C21 steroids with a 17, 20beta-dihydroxyl configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

A radioimmunoassay (RIA) has been developed to detect a range of C21 (pregnane) steroids with a 17,20beta-dihydroxyl (17,20beta) configuration. In conjunction with reverse-phase high-performance liquid chromatography (HPLC), it identifies and quantifies the metabolites of 17,20beta-dihydroxy-4-pregnen-3-one, the putative "maturation-inducing steroid" in female plaice Pleuronectes platessa. Total levels of 17,20beta metabolites which can be extracted from plasma or urine with diethyl ether (i.e., free steroids) are very low (<3 ng/ml). However, total levels of 17,20beta metabolites which can be released by solvolysis (i.e., sulphated steroids) are very high (up to 1 microg/ml in plasma and 10 microg/ml in urine). On HPLC, these sulphated metabolites have been identified (in order of abundance in plasma) as: 5beta-pregnane-3alpha,17,20beta-triol, 5beta-pregnane-3beta,17,20beta-triol, 17, 20beta-dihydroxy-4-pregnen-3-one, and 17, 20beta-dihydroxy-5beta-pregnan-3-one. These steroids are absent from plasmas of fish which have not yet begun final oocyte maturation. The results support the hypothesis that 17, 20beta-dihydroxy-4-pregnen-3-one is the maturation-inducing steroid in plaice but that it is rapidly metabolised to render it inactive. The results also show that the '17,20beta'-RIA, in combination with an overnight acid solvolysis procedure, is a useful procedure for monitoring the effects of exogenous factors (such as gonadotrophin injections) on final oocyte maturation in female plaice.

NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast.

NAP1 is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. We have examined the ability of the yeast mitotic cyclin Clb2 to function in cells that lack NAP1. Our results demonstrate that Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1 is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. Our experiments also suggest that NAP1 is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1 plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1 is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin-dependent kinase complexes induce specific events during the cell cycle.

A cell cycle checkpoint monitors cell morphogenesis in budding yeast.

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.