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.     

Presence of microtubules in isolated cortices of prophase I and metaphase II oocytes in Xenopus laevis

Summary An extensive array of microtubules has been shown to exist in the cortex of Xenopus laevis oocytes both at the prophase I and metaphase II stages. The cortical microtubules were visualized after the oocyte cortex was squashed and immunostained using anti-tubulin antibody. They are cold- and nocodazole-sensitive; their stability to both treatments decreases after meiotic maturation. Biochemical extraction of manually isolated oocyte cortices, in a microtubule-stabilizing buffer, confirms these cytological observations.     

Activation of p34cdc2 kinase by cyclin is negatively regulated by cyclic amp-dependent protein kinase in Xenopus oocytes.

Microinjection of a bacterially expressed stable delta 90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34cdc2. Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), delta 90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When delta 90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34cdc2 complex which remains inactive and tyrosine phosphorylated.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.     

Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.     

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk)

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.     

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.     

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.     

Purification and characterization of a casein-kinase-II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.     

In vitro inhibition of tubulin assembly by a ribonucleoprotein complex associated with the free ribosome fraction isolated from Xenopus laevis oocytes: effect at the level of microtubule-associated proteins

The 100 000 X g supernatant prepared from defolliculated Xenopus laevis oocytes inhibits, in a dose-dependent manner, the in vitro polymerization of rat brain neurotubulin. The oocyte inhibitory factor is thermolabile, totally inactivated by RNAse and partially by trypsin. A preliminary purification of the inhibitor showed that it is associated with the free oocyte ribosomes (80 S). The saturable binding of microtubule-associated proteins, essentially MAP2, to this 80 S ribonucleoprotein fraction is responsible for the inhibition of the in vitro tubulin assembly.     

At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes (the MPM-2 epitope), are associated with various mitotically important structures. The central mitotic regulator cdc2 kinase has been proposed to induce M-phase by phosphorylating many proteins which might include the MPM-2 antigens. To clarify the relationship of cdc2 kinase and the MPM-2 antigens, we developed an in vitro assay that enabled us to specifically detect the kinases that phosphorylate the MPM-2 epitope (ME kinases) in crude cell extracts. Two different ME kinase activities were identified in unfertilized Xenopus eggs, neither of which was cdc2 kinase, but both appeared to be activated by the introduction of cdc2 kinase into oocytes or oocyte extract. The two ME kinases differed in molecular size, substrate specificity, peptide components, and MPM-2 reactivity. The larger one, ME kinase-H, phosphorylated several MPM-2 antigens, while the smaller one, ME kinase-L, phosphorylated mainly one. We purified ME kinase-L to near homogeneity by sequential chromatography and showed that it has the characteristics of the 42-kD microtubule-associated protein (MAP) kinase. Our results support the previous finding that MAP kinase is activated during Xenopus oocyte maturation and suggest that MAP kinase may contribute to oocyte maturation induction by phosphorylating one subtype of MPM-2 epitope.     

Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase

Meiotic maturation of mammalian oocytes is under the control of cell cycle molecules Cdc2 kinase and MAP kinase (mitogen-activated protein kinase). In the present study, we investigated the relationship between the ability to activate Cdc2 kinase and MAP kinase and the acquisition of meiotic competence during pig oocyte growth. Growing and fully grown pig oocytes were collected from four groups of antral follicles of various diameters (A, 0.5-0.7 mm; B, 1.0-1.5 mm; C, 2.0-2.5 mm; D, 4.0-6.0 mm) and cultured in vitro. Fully grown oocytes from class D follicles, which have full competence to mature to metaphase II, had the ability to activate both Cdc2 kinase and MAP kinase. In contrast, growing oocytes from class A follicles, which have limited competence to resume meiosis, had no such ability. Cyclin B1 molecules did accumulate, however, with phosphorylated 35 and 36 kDa bands of p34cdc2 appearing in the cultured oocytes. Of the growing oocytes from class B follicles, 60% resumed meiosis but arrested at metaphase I. Some of the oocytes in this class were capable of activating Cdc2 kinase, although they did not appear to have established a MAP kinase-activating pathway or the ability to activate MEK. These results suggest that limited meiotic competence in growing oocytes from class A follicles is due to their inability to activate Cdc2 kinase and their incomplete MEK-MAP-kinase pathway, although the oocytes are capable of accumulating cyclin B1 molecules. During the final growth phase, pig oocytes acquire the ability to activate Cdc2 kinase and then establish the MEK-MAP-kinase pathway for full meiotic competence.     

Protein phosphatase 2A from Xenopus oocytes. Characterization during meiotic cell division.

A polyclonal antibody was raised against bacterially produced catalytic alpha subunit of protein phosphatase 2A (PP2AC) cloned from Xenopus ovarian library. The amount of PP2AC in Xenopus oocytes determined by Western blot analysis was 1 ng/microgram of cytosolic protein. The antibody depleted PP2AC from oocyte extracts in association with 6 components (40, 62, 65, 80, 85 and 90 kDa). Prophase- and metaphase-arrested oocytes contained identical amounts of PP2AC. Metaphase oocytes showed one specific change in the 62 kDa protein associated with PP2AC.     

Nucleotide torsional flexibility in solution and the use of the lanthanides as nuclear-magnetic-resonance conformation probes. The case of adenosine 5''-monophosphate

Incubation of brain extracts in the presence of 1 mM CaCl2 results in the permanent loss of tubulin polymerization, even after later addition of ethyleneglycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), when assembly conditions are chosen which rely on the presence of microtubule-associated proteins (such as MAP1 and MAP2). Purified microtubular protein, by contrast, recovers readily from calcium inhibition by the later addition of EGTA. Mixing experiments, using purified microtubular protein and brain extract, show that permanent loss of tubulin assembly is always accompanied by proteolysis of high-molecular-weight microtubular-associated proteins. Addition of purified protein MAP2 after chelation of calcium by EGTA, immediately restores microtubule assembly. Furthermore, substitution of guanosine 5'-[alpha, beta-methylene]triphosphate for GTP after EGTA treatment results in the typical tubulin polymerization process, which is independent of the presence of microtubule-associated proteins. Thus, the proteolytic action of a calcium-dependent protease is specific for high-molecular-weight microtubule-associated proteins and not tubulin itself. The protease is soluble and therefore removing during the purification of microtubular protein by cycles of temperature-dependent polymerization and depolymerization. We discuss the potential physiological importance of this calcium-dependent protease.     

Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study

Xenopus oocytes were stained by anti-tubulin and anti-MAP1 antibodies during the first meiotic cell division. In the prophase-blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti-MAP1 antibody and polyclonal anti-centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone-induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti-MAP1 and anti-centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium- and antimitotic drug sensitive. Anti-MAP1 and anti-tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti-centrosome antibody.     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA)

In Arenicola marina (Annelida, Polychaeta) the oocytes are arrested in the first prophase stage of meiosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes reach the first metaphase at which they remain arrested until fertilization. The importance of calcium in oocyte maturation has been investigated by using different drugs known to act on membrane calcium permeability and to modify intracellular free calcium concentration. Tetracaine, procaine, D-600, verapamil (Isoptin), propranolol, oxprenolol and lanthanum chloride, calcium deprivation but not ionophore A23187, are all able to induce oocyte maturation. This suggests that the brain hormone may act on the oocyte by regulating, probably increasing, the intracellular free calcium concentration, as it has been proposed for oocytes of other animals. The importance of -SH/-SS- in meiosis reinitiation is suggested by the fact that dithiothreitol and 2, 3-dimercaptopropanol, two disulfide reducing agents, both induce oocyte maturation.