Characterization and expression of a maternal axolotl cyclin B1 during oogenesis and early development.

The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr15), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr15 cyclic accumulations are observed with kinetics different from those of the early embryonic cycles. The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.

Activation of maturation promoting factor in Bufo arenarum oocytes: injection of mature cytoplasm and germinal vesicle contents.

Although progesterone is the established maturation inducer in amphibians, Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured. Reinitiation of meiosis is regulated by maturation promoting factor (MPF), a complex of the cyclin-dependent kinase p34cdc2 and cyclin B. Although the function and molecule of MPF are common among species, the formation and activation mechanisms of MPF differ according to species. This study was undertaken to evaluate the presence of pre-MPF in Bufo arenarum oocytes incompetent to mature spontaneously and the effect of the injection of mature cytoplasm or germinal vesicle contents on the resumption of meiosis. The results of our treatment of Bufo arenarum immature oocytes incompetent to mature spontaneously with sodium metavanadate (NaVO3) and dexamethasone (DEX) indicates that these oocytes have a pre-MPF, which activates and induces germinal vesicle breakdown (GVBD) by dephosphorylation on Thr-14/Tyr-15 by cdc25 phosphatase and without cyclin B synthesis. The injection of cytoplasm containing active MPF is sufficient to activate an amplification loop that requires the activation of cdc25 and protein kinase C, the decrease in cAMP levels, and is independent of protein synthesis. However, the injection of germinal vesicle content also induces GVBD in the immature receptor oocyte, a process dependent on protein synthesis but not on cdc25 phosphatase or PKC activity.

Genistein blocks breast cancer cells in the G(2)M phase of the cell cycle.

Genistein, a natural isoflavone phytoestrogen present in soybeans, caused a dose-dependent growth inhibition of the two hormone-sensitive cell lines T47D and ZR75.1 and of the two hormone-independent cell lines MDAMB-231 and BT20. Flow cytometric analysis of cells treated for 4 days with 15 and 30 microM genistein showed a dose-dependent accumulation in the G(2)M phase of the cell cycle. At the highest tested concentration, there was a sevenfold increase in the percentage of cells in G(2)M (63%) with respect to the control (9%) in the case of T47D cells and a 2.4-fold increase in the case of BT20. An intermediate fourfold accumulation was observed in the case of MDAMB-231 and ZR75.1. The G(2)M arrest was coupled with a parallel depletion of the G(0)/G(1) phase. To understand the mechanism of action underlying the block in G(2)M induced by genistein, we investigated the expression and the activity of cyclins and of cyclin-dependent kinases specifically involved in the G(2)-->M transition. As expected, p34(cdc-2) expression, monitored by Western blotting, was unaffected by genistein treatment in all cell lines. With exception of the T47D cell line, we revealed an increase in the tyrosine phosphorylated form of p34, suggesting an inactivation of the p34(cdc-2) catalytic activity consequent to treatment of cells with genistein. In fact, immunoprecipitates from genistein-treated MDAMB-231 and BT20 cells displayed a fourfold decrease in kinase activity evaluated using the histone H1 as substrate. Conversely, no variation in kinase activity was observed between treated and untreated ZR75.1 cells despite the increase in p34 phosphorylation. In cells treated with 30 microM genistein, cyclin B(1) (p62) increased 2.8-,8-and 103-fold, respectively, in BT20, MDAMB-231, and ZR75.1 cells, suggesting an accumulation of the p62, which is instead rapidly degraded in cycling cells. No effects were observed on cyclin expression in T47D cells. We therefore conclude that genistein causes a G(2)M arrest in breast cancer cell lines, but that such growth arrest is not necessarily coupled with deregulation of the p34(cdc-2)/cyclin B(1) complex only in all of the studied cell lines.

Partial purification of maturation-promoting factor from catfish, Clarias batrachus: identification as the histone H1 kinase and its periodic activation.

Maturation-promoting factor (MPF) has been demonstrated in the 100,000 g supernatant of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP)-induced catfish, Clarias batrachus oocytes using DEAE-cellulose and sephadex G-200 chromatography. Partially purified MPF molecule eluted as a single peak on sephadex G-200 with molecular mass of approximately 200 kDa in native PAGE. SDS-PAGE analysis showed the presence of five proteins of 32, 34, 45, 46 and 48 kDa. Antibody against the PSTAIR sequence of p34cdc2 recognized 32 and 34 kDa proteins, whereas rabbit anti-cyclin B1 and B2 crossreacted with 46 and 48 kDa proteins, respectively. Cyclin B was absent in immature oocytes and appeared after 7 h of 17 alpha, 20 beta-DP stimulation, coinciding with the histone H1 kinase (HH1K) activity and start of germinal vesicle breakdown (GVBD). Our data indicate that C. batrachus MPF is a complex of cdc2 kinase and cyclin B molecules. A close relationship between HH1K activity and catfish oocyte maturation has been demonstrated using cycloheximide, cytochalasin B and colchicine. HH1K activation was inhibited by cycloheximide, while cytochalasin B and colchicine were ineffective. These finding suggests that the activation of HH1K depends on protein synthesis, whereas disruption of microfilaments influences only nucleus migration without effect on GVBD or HH1K activation. An increase of phosphorylated proteins after activation of catfish oocytes with 17 alpha, 20 beta-DP has also been observed.

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.

Purification and characterization of a p13suc1-bound serine/threonine kinase that is expressed in mature rat brain.

We previously reported that the histone-H1 kinase activity bound to p13suc1 increased dramatically during development of the rat brain. In the present work, an in situ kinase assay in an SDS/polyacrylamide gel that contained substrate proteins was employed to characterize the enzyme. Two major proteins of 45 kDa and 100 kDa were found to have p13suc1-bound histone-H1 kinase activity. The former (p45) exhibited strong activity towards histone H1 and had weak autophosphorylation activity, whereas the latter (p100) acted on myelin basic protein or histone H1, and underwent autophosphorylation. p45 was further purified from the nuclear-enriched fraction of rat brain to near homogeneity through sequential column chromatographies. The purified enzyme retained its ability to bind specifically to p13suc1, which suggests that this binding does not require a cofactor. The immunochemical and enzymatic properties of p45 revealed that it differs from Cdk that are known to bind to p13suc1 with high affinity. However, in vitro p45 acted on the peptide motif that is conserved among substrates for cyclin-dependent kinases (Cdk) and mitogen-activated protein kinases, which implies that this protein might belong to the large family of proline-directed kinases. The evidence obtained in this study suggest that p45 is a nuclear p13suc1-bound kinase that has unique functions in the mature brain.

Behavior of the components of maturation-promoting factor, cdc2 kinase and cyclin B, during oocyte maturation of goldfish.

We examined the changes that occurred in the two components of maturation-promoting factor (MPF), cdc2 kinase and cyclin B, during oocyte maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13suc1 beads and anticyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2 micrograms/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.

Cyclin synthesis: who needs it?

Studies of the G2 to M transition in amphibian oocytes, in combination with in vitro mitotic systems and yeast genetic analysis, have significantly contributed to our understanding of the mechanisms by which M-phase is regulated. Historically, oocyte maturation has provided a number of valuable initial observations, but the biochemical elucidation of cell cycle control mechanisms has proved more tractable in cell-free extracts of frog eggs which reproduce aspects of early embryogenic mitosis. Recent experiments examining the importance of protein synthesis in the maturing oocyte have highlighted some important differences between mitosis and meiosis. Additional controls found in meiosis but not embryonic mitosis, are similar to controls found in somatic cells. This suggests that understanding the differences, as well as the similarities, between meiosis in the oocyte and mitosis in the early embryo will help us to learn more about the way in which cells enter and leave mitosis.

Purification and characterization of maturation-promoting factor in fish.

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.

Histone H1 kinase specific to the SPKK motif.

A protein kinase phosphorylating sea urchin spermatogenous histones, H1 and H2B, was found in sea urchin egg homogenate and purified. The kinase is activated by cAMP and is composed of two different types of subunits with molecular masses 41 and 46 kDa. The kinase phosphorylates a peptide, Ser-Pro-Arg-Lys-Ser-Pro-Arg-Lys, which is a double repeat of the DNA-binding SPKK motif [Suzuki M., (1989) EMBO J. 8, 797-804]. We name this kinase SPkinase because it exclusively phosphorylates H1 and H2B, the only histones containing SPKK motifs. Phosphorylation of H1 by SPkinase decreases the DNA-binding ability of H1. This paper is the first to report purification of a kinase which affects the DNA-binding ability of a gene regulatory protein.