UV irradiation of pig metaphase chromosomes: maturation-promoting factor degradation, nuclear cytology and cell cycle progression

Experiments were designed to test two hypotheses. The first was that irradiation of pig metaphase chromosomes would block the normal sequence of cytological and molecular events associated with activation; the second postulated that damaged DNA would prevent eggs from progressing through the first mitotic cleavage cycle. The experimental protocol involved selectively irradiating the metaphase II plate of pig oocytes with highly focused 254 nm ultraviolet (UV) light, followed by activation using standard electroactivation procedures. The following assessments were made of different groups of eggs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) changes in maturation-promoting factor kinase (MPF kinase) activity at 1 h intervals after activation; and (iv) mitotic progression of eggs containing damaged chromosomal fragments. UV irradiation neither prevented the reassembly of nuclear membranes required for pronuclear formation nor interfered with the normal pattern of MPF kinase degradation after egg activation. UV irradiation did induce a wide range of chromatin defects, including condensation and dispersal of DNA fragments which, in turn, resulted in the formation of micronuclei in the treated eggs and embryos. The presence of damaged DNA retarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely affected by the presence of UV-damaged DNA. Overall, these results indicate that early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. In this respect, pig eggs are similar to Xenopus eggs, but are different from bovine eggs. On the basis of these findings it is suggested that focused UV irradiation offers a simple and rapid technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental stages.     

Caulerpenyne blocks MBP kinase activation controlling mitosis in sea urchin eggs

In a previous study, we demonstrated that caulerpenyne (Cyn), a natural sesquiterpene having an antiproliferative potency, blocked the mitotic cycle of sea urchin embryos at metaphase and inhibited the phosphorylation of several proteins, but did not affect histone H1 kinase activation (Pesando et al, 1998, Eur. J. Cell Biol. 77, 19-26). Here, we show that concentrations of Cyn that blocked the first division of the sea urchin Paracentrotus lividus embryos in a metaphase-like stage (45 microM) also inhibited the stimulation of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MBPK). However, Cyn had no effect on MBP phosphorylation when added in vitro to an untreated egg extract taken at the time of metaphase, suggesting that Cyn acts on an upstream activation process. PD 98059 (40 microM), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea urchin eggs at metaphase in a way very similar to Cyn. Both molecules induced similar inhibitory effects on MBP kinase activation in vivo, but had no direct effect on MBP kinase activity in vitro, whereas they did not affect H1 kinase activation neither in vivo nor in vitro. As a comparison, butyrolactone 1 (100 microM), a known inhibitor of H1 kinase activity, did inhibit H1 kinase of sea urchin eggs in vivo and in vitro, and blocked the sea urchin embryo mitotic cycle much before metaphase. Immunoblots of mitotic extracts, treated with anti-active MAP-kinase antibody, showed that both Cyn and PD 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MBPK activation pathway. They also show that H1 kinase and MBPK activation can be dissociated from each other in this model system.     

M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis

Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation. H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity. H1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation. Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K. These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation.     

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.     

Differential regulation of cyclin B1 degradation between the first and second meiotic divisions of bovine oocytes

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 μM, but higher doses (200 μM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.     

Activation of myelin basic protein kinases during echinoderm oocyte maturation and egg fertilization

At least five activated protein kinases were detectable in soluble extracts from maturing as compared to immature sea star oocytes. These kinases could be distinguished on the basis of the time courses of their activation following exposure of the oocytes to 1-methyladenine, their substrate specificities, and their chromatographic properties on DEAE-Sephacel and Sephacryl S-200. A histone H1 kinase (HH1K) (Mr 110,000) underwent maximal activation near the time of 1-methyladenine-induced germinal vesicle breakdown (GVBD). When myelin basic protein (MBP) was used as a substrate, HH1K and two additional kinases (MBPK-I and MBPK-II) were detectable. MBPK-II (Mr 110,000) was fully activated at the time of GVBD, whereas peak activation of MBPK-I (Mr 45,000) occurred after this event. Two "ribosomal protein S6 kinases" (S6K-I and S6K-II) could be detected with a synthetic peptide (RRLSSLRA), which was patterned after a major phosphorylation site in S6. The two S6 kinases (Mr 110,000 for both) underwent activation post-GVBD. HH1K and S6K-I coeluted from DEAE-Sephacel at a conductivity of 5.5-6.0 mmho, whereas MBPK-I, MBPK-II, and S6K-II coeluted from this resin in a second peak at a conductivity = 10-11 mmho. The HH1K and MBPK-II activities both declined prior to the emission of the first polar body (i.e., meiotic cell division), but the MBPK-I, S6K-I, and S6K-II activities remained elevated during this time. The activities of these kinases were also examined during the early cell divisions in sea urchin embryos. Within 5 min after fertilization, the high level of MBPK-I activity in sea urchin eggs rapidly declined. However, along with the HH1K and MBPK-II activities, the MBPK-I activity was transiently increased prior to each cell division. No appreciable postfertilization changes in the S6K-I and S6K-II activities were apparent during the first three cycles of cell division.     

Modulation of cell cycle control during oocyte‐to‐embryo transitions

Ex ovo omnia—all animals come from eggs—this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII‐arrest), the first mitotic cell cycle, and early embryonic divisions.     

Migration and division of cleavage nuclei in the gall midge,Wachtliella persicariae

Summary The control of nuclear division and migration was studied in time-lapse films of the multinucleate egg cell of a gall midge by experimental alterations of the mitotic pattern. During each cleavage cycle, a wave of randomly oriented saltations of yolk particles (WROS) is seen to travel through the ooplasm. This wave proved to be an indispensable prerequisite for the accompanying anaphase wave and for the activation of the nuclear migration cytasters: WROS cycles can occur autonomously without cleavage nuclei being present, but there is no anaphase without a WROS passing the dividing nucleus. WROSs and mitotic waves can be inverted, and the WROS cycles and the cleavage cycles can be desynchronized by temperature grandients or by locally impaired gas exchange. If a nucleus is not ready for anaphase when met by a WROS, it will only divide in the course of the next WROS. WROSs thus indicate autonomous anaphase-triggering waves governing the cleavage divisions. Rhythmic ooplasmic movements continue even if the WROSs as well as the nuclear divisions are inhibited by colchinine. The characteristics of the WROSs support the hypothesis that each of them is the visible effect of a wave of calcium release (similar to that established in vertebrate eggs) which acts locally on the microtubular system and may continue even if the WROSs are suppressed. The correlations between a possible calcium release, WROS activity, microtubule disassembly and nuclear cycle are discussed.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Studies of Unequal Cleavage in Molluscs: I. Nuclear Behavior and Anchorage of a Spindle Pole to Cortex as Revealed by Isolation Technique

Unequal division of the eggs of Spisula solidissima was studied by isolating the nuclear elements. The isolation technique consists mainly of the use of Na-lauryl sulphate with occasional inverting of the container of alcohol-fixed eggs. The procedure was applied to the following stages. (1) germinal vesicles, (2) 1st maturation division, showing anchorage of a spindle pole to the cortex, (3) 2nd meiosis, (4) pronuclear encouter, (5) metaphase, (6) shifting of the metaphase spindle to one side of the cell and a contact with the cortex, (7) ana- and telophase of the 1st division.
Migration and "anchorage" of the spindle to the cortex by one pole is a common phenomenon in unequal divisions of polar body formation and of egg cleavage.     

Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs

To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca²⁺ and exocytosis of cortical alveoli (CABD). The sperm‐penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although it decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at ≤40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6‐dimethylaminopurine (6‐DMAP), however, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short‐lived protein kinase(s), sensitive to 6‐DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Dev. Genet. 25:137–145, 1999. © 1999 Wiley‐Liss, Inc.     

Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.     

Nuclear division and migration during early embryogenesis of Bradysia tritici coquillet (syn. Sciara ocellaris) (diptera : Sciaridae)

Nuclear division and migration of cleavage nuclei in the embryos of Bradysia tritici (Diptera : Sciaridae) have been studied by light microscopy and nuclear staining. There are 8 cleavage cycles up to the syncytial blastoderm stage (4.5 hr), and during the 11th cycle cellularization begins (6.5 hr). The first 3 divisions take about 30 min each. During the 5th and 6th cycles, the maximum rate of division is reached (12 min/cycle at 22°C). After pole cell formation, the duration of the following mitotic cycles increases progressively. During nuclear migration, the presumptive germ line nuclei reach the egg cortex first, followed by anterior somatic nuclei and finally, posterior somatic nuclei reach the egg cortex. Possibly as a result of this region-specific nuclear migration, nuclear divisions become parasynchronous after 3 hr of embryogenesis (4th cycle). Several mitotic cycles later, between the 8th and 10th cycle in different embryos, X-chromosome elimination in somatic nuclei begins at the anterior egg pole and progresses in anteroposterior direction. Our observations suggest that the observed region-specific differences may be due to the activity of localized factors in the egg that control migration and nuclear cycle of the somatic nuclei.     

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C¹⁴-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.     

Transient reactivation of CSF in parthenogenetic one-cell mouse embryos

During meiosis, the cytostatic factor (CSF) activity stabilizes the activity of the M-phase promoting factor (MPF) in metaphase II arrested vertebrate oocytes. Upon oocyte activation, the inactivation of both MPF and CSF enables the entry into the first embryonic mitotic cell cycle. Using a biological assay based on cell-fusion (hybrid between a parthenogenetically activated egg entering the first mitotic division and an activated oocyte), we observed that in activated mouse oocytes a first drop in CSF activity is detectable as early as 20 min post-activation. This suggests that CSF is inactivated upon MPF inactivation. However, CSF activity increases again to reach a maximum 60 min post-activation and gradually disappears during the following 40 min. Thus, in activated mouse oocytes (undergoing the transition to interphase) CSF activity fluctuates before definitive inactivation. We found that hybrids arrested in M-phase, thus containing CSF activity after oocyte activation, have activated forms of MAP kinases while hybrids in interphase have inactive forms of these enzymes. We postulate that CSF inactivation in mouse oocytes proceeds in two steps. The initial inactivation of CSF, required for MPF inactivation, is transient and does not require MAP kinase inactivation. The final inactivation of CSF, required for normal embryonic cell cycle progression, is dependent upon the inactivation of MAP kinases.     

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.     

Characterization of protein kinase C-delta in mouse oocytes throughout meiotic maturation and following egg activation

Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-delta), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-delta, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-delta distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-delta became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-delta is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.     

Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase

In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30 min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.     

Translation of incenp during oocyte maturation is required for embryonic development in Xenopus laevis

The chromosome passenger complex (CPC) consists of Aurora-B kinase and several other subunits. One of these, incenp, binds Aurora-B and regulates its kinase activity. During Xenopus oocyte maturation, incenp accumulates through translation, contributing to aurora-b activation. A previous study has demonstrated that inhibition of incenp translation during oocyte maturation diminishes aurora-b activation but does not interfere with oocyte maturation, characterized by normal maturation-specific cyclin-b phosphorylation, degradation, and resynthesis. Here we have extended these findings, showing that inhibition of incenp translation during oocyte maturation did not interfere with meiosis I or II, as indicated by the normal emission of the first polar body and metaphase II arrest, followed by the successful emission of the second polar body upon parthenogenetic egg activation. Most importantly, however, when transferred to host frogs and subsequently ovulated, the incenp-deficient eggs were fertilized but failed to undergo mitotic cleavage. Thus, translation of incenp during oocyte maturation appears to be part of oocyte cytoplasmic maturation, preparing the egg for the rapid mitosis following fertilization.     

Oocyte activation and passage through the metaphase/anaphase transition of the meiotic cell cycle is blocked in clams by inhibitors of HMG-CoA reductase activity

Cell cycle progression for postembryonic cells requires the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), the enzyme which catalyzes the production of the isoprenoid precursor, mevalonate. In this study, we examine the requirements of HMG-R activity for cell cycle progression during the meiotic and early mitotic divisions using oocytes and dividing embryos from the surf clam, Spisula solidissima. Using two different inhibitors of HMG-R, we find that the activity of this enzyme appears to be required at three distinct points of the cell cycle during meiosis. Depending on the stage at which these inhibitors are added to synchronous clam cultures, a reversible cell cycle block is triggered at the time of activation or at metaphase of either meiosis I or II, whereas there is not block to the mitotic cell cycle. Inhibition of HMG-R activity in activated oocytes does not affect the transient activation of p42MAPK but results in a block at metaphase of meiosis I that is accompanied by the stabilization of cyclins A and B and p34cdc2 kinase activity. Our results suggest that metabolites from the mevalonate biosynthetic pathway can act to influence the process of activation, as well as the events later in the cell cycle that lead to cyclin proteolysis and the exit from M phase during clam meiosis.