Time-related effect of GnRH on histone H1 kinase activity in the goldfish follicle-enclosed oocyte

The level of cyclin B-associated cdc2 kinase, a component of maturation promoting factor (MPF), is known to be high during metaphase of the meiotic maturation of oocytes. The time-related action of gonadotropin-releasing hormones (GnRH) on histone H1 kinase activity (known to reflect cdc2 kinase activity) was investigated in vitro in follicle-enclosed goldfish oocytes. Germinal vesicle breakdown (GVBD) and testosterone production were also investigated in the same follicle-enclosed goldfish oocytes to determine the temporal relationship between GnRH-induced histone H1 kinase activity and the reinitiation of meiosis and steroidogenesis. Treatments with gonadotropin (GTH) or GnRH stimulated the histone H1 kinase activity to the same maximum level. However, sGnRH- and cGnRH-II-induced histone H1 kinase activity were significantly higher compared with controls after 2 hours of treatment, whereas the GTH-induced increase became significantly higher after 6-8 hours of incubation. Overall, the results demonstrate a close temporal relationship between GVBD response and histone H1 kinase activity induced by GTH and sGnRH-cGnRH-II.     

Studies on the mechanism of activation of mitotic histone H1 kinase

A chromatin-associated histone H1 kinase has been detected in synchronized Novikoff hepatoma cells. Enzyme specific activity increased 4 to 6-fold from late G-2 to mid-metaphase, then decayed exponentially ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 28.5 min) to the interphase level. Extracts of the mitotic kinase retained the ability to decay $\text{in}\text{vitro}$ at 37°C but not at 0°C ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 24 min), under conditions in which interphase activity was stable. Sedimentation rates in sucrose density gradients of interphase and mitotic enzymes (before and after decay) were identical. Purification did not alter the rate of enzyme decay. However, high ionic strength prevented decay of crude but not purified preparations of mitotic enzyme. The results are discussed in terms of an allosteric mechanism for reversible activation of enzyme activity.     

Autonomous activation of histone H1 kinase, cortical activity and microtubule organization in one- and two-cell mouse embryos

The activation of M-phase promoting factor (MPF) in one-cell mouse embryo is independent from the nucleus. Other autonomous phenomena include the cortical activity observed at the end of the first cell cycle and the reorganization of the microtubule network. Here, we observed that the autonomous control of MPF activation is present also in two-cell mouse embryos (H1 kinase activity being higher in the first than in the second cell cycle). Moreover, the disappearance of the cortical activity in anucleated halves is observed when MPF activation takes place. The rounding up of the cytoplast and the mitotic reorganization of the microtubule network correlates with the maximum activity of H1 kinase in anucleated halves from one-cell embryos. In anucleated halves of two-cell stage blastomeres neither the cortical activity nor the microtubule reorganization were observed. The degree of activation of histone H1 kinase, and, as a consequence, the cortical activity and the microtubule reorganization, does not depend on the distribution of cyclin B. Finally, the level of cyclin B synthesis is similar in anucleated and nucleated halves derived from both one- and two-cell embryos.     

Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions

Fertilized sea urchin eggs undergo a series of rapid and synchronized mitotic divisions. Extracts were made at various times throughout the first three mitotic divisions and assayed for phosphorylating activity toward histone H1. Histone H1 kinase (HH1K) undergoes a transient activation (8- to 10-fold increase) 20 min before each cleavage. The amplitude of the HH1K peak strongly depends on the synchrony of the egg population. Concomitant cytological observations show that the time-course of HH1K correlates with the time-course of nuclear envelope breakdown and of metaphase. This correlation is observed at each cell division cycle. HH1K from each of the three first mitoses show identical time- and concentration-dependence curves as well as identical dose-inhibition curves with 6-dimethylaminopurine and quercetin, suggesting that the same (group of) kinase(s) is (are) activated before each cleavage. Ionophore A23187 does not trigger, but inhibits, HH1K activation; however, partial activation of the eggs with ammonia at pH 9.0 (but not at pH 8.0) triggers the transient HH1K activation. Appearance of the HH1K cycle requires protein synthesis since it is completely abolished in emetine-treated eggs. Although cytochalasin B blocks egg cleavage, it does not inhibit HH1K activation nor nuclear divisions. A prolonged HH1K activation cycle is observed in eggs arrested in metaphase with colchicine or nocodazole. Despite the existence of a cycle in cAMP concentration during mitosis, forskolin, an activator of adenylate cyclase, does not modify the time-course of HH1K activation and of cell division. The cycling HH1K is independent of calcium-calmodulin, calcium-phospholipids, or cyclic AMP. It clearly resembles the mammalian "growth-associated histone kinase." The relationship between the transient activation of HH1K and the intracellular mitotic factors driving the cell cycle is discussed.     

Protein phosphatases possibly involved in rat spermatogenesis

The expression of mRNAs for catalytic subunits of serine/threonine protein phosphatases 1 (PP-1) and 2A (PP-2A) in various rat tissues were examined. Four kinds of probes were used to detect mRNAs for two isotypes of PP-1 (dis2m1 and dis2m2), and two isotypes of PP-2A (PP-2A alpha and PP-2A beta). mRNAs for all of these four catalytic subunits were expressed in almost all tissues at substantial levels. They were expressed in two different sizes in most tissues. Remarkable evidence is that the smaller sized mRNAs of dis2m1 and PP-2A beta, 1.8 kb and 1.4 kb in length, respectively, were specifically highly expressed in testis. Both these smaller sized mRNAs began to be expressed at the age when meiosis started and were detected in testicular germ cells at the pachytene stage of meiotic prophase. Protein phosphatases which have peptides encoded by dis2m1 and PP-2A beta as catalytic subunits may play important roles in spermatogenesis.     

AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro

We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo.     

Purification and characterization of a specific histone H1 protein kinase from mouse plasmacytoma

A protein kinase with high specificity for histone H1 was purified from a plasmacytoma microsomal fraction by a high-salt wash, ammonium sulfate precipitation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-200 columns, and the main properties of this kinase were studied. A sulfhydryl compound, such as 2-mercaptoethanol or dithiothreitol, was necessary for full activity. The optimum pH was 7.4-7.8. After purification, the histone H1 kinase was not stimulated by cAMP or cGMP. It was not inhibited by the heat-stable cAMP-dependent protein kinase inhibitor from beef heart. It utilized preferentially GTP over ATP as phosphate donor. Km values for ATP and GTP were 58 microM and 1.4 microM respectively; the Km for histone H1 was 14 microgram ml-1. The molecular weight was approximately 90 000 by gel-exclusion chromatography. Analysis of the purified H1-specific protein kinase by polyacrylamide gel electrophoresis in dodecylsulfate showed two bands having molecular weights of approximately 64 000 and 54 000. Many characteristics of this kinase were similar to those of the chromatin-bound protein kinase reported by other workers in rapidly proliferating cells.     

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.     

Changes in histone acetylation during mouse oocyte meiosis

We examined global changes in the acetylation of histones in mouse oocytes during meiosis. Immunocytochemistry with specific antibodies against various acetylated lysine residues on histones H3 and H4 showed that acetylation of all the lysines decreased to undetectable or negligible levels in the oocytes during meiosis, whereas most of these lysines were acetylated during mitosis in preimplantation embryos and somatic cells. When the somatic cell nuclei were transferred into enucleated oocytes, the acetylation of lysines decreased markedly. This type of deacetylation was inhibited by trichostatin A, a specific inhibitor of histone deacetylase (HDAC), thereby indicating that HDAC is able to deacetylate histones during meiosis but not during mitosis. Meiosis-specific deacetylation may be a consequence of the accessibility of HDAC1 to the chromosome, because HDAC1 colocalized with the chromosome during meiosis but not during mitosis. As histone acetylation is thought to play a role in propagating the gene expression pattern to the descendent generation during mitosis, and the gene expression pattern of differentiated oocytes is reprogrammed during meiosis to allow the initiation of a new program by totipotent zygotes of the next generation, our results suggest that the oocyte cytoplasm initializes a program of gene expression by deacetylating histones.     

Protein tyrosine phosphatases in Chaetopterus egg activation

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPsigma, -rho, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with beta-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately 140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.     

Accumulation of the isolated carboxy-terminal domain of histone H1 in the Xenopus oocyte nucleus

Histone H1 accumulates in the nucleus after injection into the cytoplasm of Xenopus oocytes. A proteolytic fragment of 89 amino acids encompassing the carboxy-terminal domain also accumulates in the nucleus. Lysine, alanine and proline compose 84% of this domain. Accumulation is not due solely to the high lysine content since poly-L-lysine does not accumulate in the nucleus when injected into the cytoplasm of Xenopus oocytes. Proteolytic fragments encompassing other domains of the molecule are degraded in the oocyte after injection. In these instances degradation is more rapid in the cytoplasm than in the nucleus giving the false impression of accumulation in the nucleus, an artefact which is likely to confuse other studies of protein migration. Susceptibility to rapid degradation is a dominant feature, thus the globular domain destabilises the contiguous carboxy-terminal domain. The properties of the carboxy-terminal domain of H1 and the possible involvement of the amino acids lysine, proline and alanine in migration are discussed and compared with those of a domain that specifies migration of nucleoplasmin into the oocyte nucleus.     

Protein kinase C in tumoricidal activation of mouse macrophage cell lines

A potential role of protein kinase C (PKC) in lipopolysaccharide- (LPS-) induced tumoricidal activation of macrophages was investigated by using two mouse macrophage cell lines (P388D1 and J774). J774 cells are stimulated by LPS to kill target P815 mastocytoma cells, whereas P388D1 cells fail to develop such an ability. Pretreatment of J774 cells with H-7 or phorbol myristate acetate resulted in a significant inhibition of LPS-induced cytotoxicity, whereas pretreatment with H-8, ML-7, HA1004, or W-7 did not. Since these results suggested a critical role of PKC in the activation process, the properties of PKC in the two cell lines were compared. Western blotting with rabbit antiserum specific for the PKC beta regulatory domain allowed detection of a protein of 79 kilodaltons (kDa) in the detergent lysates of both cell lines that were not stimulated by LPS. However, LPS treatment resulted in the appearance of a second protein of 40 kDa only in J774 cells and not in P388D1 cells. Furthermore, two forms of protein kinase (one basic and the other acidic) were identified in the cytosol of J774 cells by HPLC on DEAE-5PW, whereas only the basic form was found in P388D1 cells. On the basis of the response of the basic and acidic form protein kinases to phosphatidylserine (PS), diolein, and Ca2+, the basic form was found to contain both regulatory and catalytic domains of PKC, whereas the acidic form was suggested to represent the PKC catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte

We have characterized plk1 in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD), is maximal at GVBD, and is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form. However, plk1 phosphorylation is inhibited after treatment with two specific p34(cdc2) inhibitors, roscovitine and butyrolactone, suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the cytoplasm and enriched in the nucleus and during prometaphase is localized to the spindle poles. At anaphase it relocates to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1 becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast migrating form with weak kinase activity is present. In this work we show that plk1 is present in mouse oocytes during meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this period suggest its implication in the organization and progression of M-phase.     

Differential phosphorylation of histone H1 subfractions in vivo.

A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles ($\text{Rana}\text{catesbeiana}$). Two H1 subfractions were obtained which differed in terms of net incorporation of [³²P]phosphate $\text{in}\text{vivo}$. Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [³²P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [³²P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues.     

In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte

We have characterized a serine/threonine protein kinase from Xenopus metaphase-II-blocked oocytes, which phosphorylates in vitro the microtubule-associated protein 2 (MAP2). The MAP2 kinase activity, undetectable in prophase oocytes, is activated during the progesterone-induced meiotic maturation (G2-M transition of the cell cycle). p-Nitrophenyl phosphate, a phosphatase inhibitor, is required to prevent spontaneous deactivation of the MAP2 kinase in crude preparations; conversely, the partially purified enzyme can be in vitro deactivated by the low-Mr polycation-stimulated (PCSL) phosphatase (also termed protein phosphatase 2A2), working as a phosphoserine/phosphothreonine-specific phosphatase and not as a phosphotyrosyl phosphatase indicating that phosphorylation of serine/threonine is necessary for its activity. S6 kinase, a protein kinase activated during oocyte maturation which phosphorylates in vitro ribosomal protein S6 and lamin C, can be deactivated in vitro by PCSL phosphatase. S6 kinase from prophase oocytes can also be activated in vitro in fractions known to contain all the factors necessary to convert pre-M-phase-promoting factor (pre-MPF) to MPF. Active MAP2 kinase can activate in vitro the inactive S6 kinase present in prophase oocytes or reactivate S6 kinase previously inactivated in vitro by PCSL phosphatase. These data are consistent with the hypothesis that the MAP2 kinase is a link of the meiosis signalling pathway and is activated by a serine/threonine kinase. This will lead to the regulation of further steps in the cell cycle, such as microtubular reorganisation and S6 kinase activation.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturation

The requirement of the germinal vesicle (GV) for the normal kinetics of mitogen-activated protein (MAP) kinase activity during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated, and the locations of their extracellular signal-regulated kinases 1 and 2 (ERK1/2), major MAP kinases in maturating porcine oocytes, were detected by indirect immunofluorescent microscopy. The MAP kinase activity was assayed as myelin basic protein (MBP) kinase activity, and the phosphorylation states of ERK1/2 were detected by immunoblotting analyses. Translocation of MAP kinase into the GV and association with the spindle were observed in intact oocytes, while MAP kinase in enucleated oocytes was distributed almost uniformly in cytoplasm throughout the culturing period. The phosphorylation and the activation of MAP kinase were induced, and the activity was comparable with that of control denuded oocytes. The high level of activity was maintained through maturation, even in the absence of spindle formation. These results indicate that the presence of nuclear material and translocation into the GV are dispensable for the activation of MAP kinase and that associating with the spindle is not required for maintenance of its activity though porcine oocyte maturation.