Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.     

In Vitro Induction of Starfish Oocyte Maturation by Cysteine Alkylesters

The effect of various disulfide-reducing agents including cysteine and its alkylesters on the induction of germinal vesicle breakdown (GVBD) in starfish ( Asterina pectinifera ) oocytes was investigated in vitro . Although cysteine did not induce GVBD, its alkylesters were effective. Cysteine alkylesters significantly mimicked the effect of 1-methyladenine (1-MeAde), the naturally occurring maturation-inducing hormone of starfish, on oocyte maturation. However, the effective concentrations and pH optimum for stimulation of oocyte maturation varied between 1-MeAde and the cysteine alkylesters. By comparing pKa values of the disulfide-reducing agents to pH of the medium, it is suggested that the redox potential of a disulfide-reducing agent is an important indicator its ability to induce oocyte maturation.
With the use of fluorescent probes for thiol groups, it was shown that the fluorescence in oocyte cortices increased within 5 min after administration of 1-MeAde. The fluorescence intensity in the cortices also increased after treatment with cysteine and its alkylesters, although the intensity was much stronger with the latter. Furthermore, both 1-MeAde and the disulfide-reducing agents were suggested to cause reduction of thiol groups within the plasma membrane as opposed to those on the external and internal surfaces. Thus, it is suggested that disulfide-reducing agents and 1-MeAde induce starfish oocyte maturation by changing the redox state of the thiol groups located within the oocyte plasma membrane.     

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro-tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes. © 1996 Wiley-Liss, Inc.     

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes

A transient increase in intracellular Ca²⁺ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca²⁺ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca²⁺ transient followed by GVBD. Even when the transient increase of Ca²⁺ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca²⁺ increase is neither necessary nor sufficient for maturation of the starfish oocyte.
When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca²⁺ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca²⁺ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

Golgi apparatus dynamics during mouse oocyte in vitro maturation: effect of the membrane trafficking inhibitor brefeldin A

We have studied Golgi apparatus dynamics during mouse oocyte in vitro maturation, employing both live imaging with the fluorescent lipid BODIPY-ceramide and immunocytochemistry using several specific markers (beta-COP, giantin, and TGN38). In germinal vesicle oocytes the Golgi consisted of a series of structures, possibly cisternal stacks, dispersed in the ooplasm, but slightly more concentrated in the interior than at the cortex. A similar pattern was detected in rhesus monkey germinal vesicle oocytes. These "mini-Golgis" were functionally active because they were reversibly disrupted by the membrane trafficking inhibitor brefeldin A. However, the drug had no visible effect if the oocytes had been previously microinjected with GTP-gamma-S. During in vitro maturation the large Golgi apparatus structures fragmented at germinal vesicle breakdown, and dispersed homogenously throughout the ooplasm, remaining in a fragmented state in metaphase-II oocytes. Similarly to what has been reported using protein synthesis inhibitors, the presence of brefeldin A blocked maturation at the germinal vesicle breakdown stage before the assembly of the metaphase-I spindle. These results suggest that progression of murine oocyte maturation may require functional membrane trafficking.     

Starfish oocyte maturation: 1-methyladenine triggers a drop of cAMP concentration related to the hormone-dependent period

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Oocytes of Evasterias troschelii contain 0.43 pmole cyclic AMP/mg protein and 0.47 pmole cyclic GMP/mg protein. Upon stimulation by 1-MeAde the oocytes undergo a moderate (10-30%) decrease in their cAMP concentration. The concentration of cGMP remains unaltered. Oocytes treated with forskolin, an activator of adenylate cyclase, increase their cAMP concentration over 35-fold, up to 16 pmole cAMP/mg protein. When stimulated by 1-MeAde these forskolin-pretreated oocytes undergo a major (50-70%) decrease in their cAMP concentration. A similar decrease is triggered by mimetics of 1-MeAde, such as dithiothreitol, arachidonic acid (AA), and 8-hydroxyeicosatetraenoic acid (8-HETE), but not by adenine which is inactive. 1-MeAde-stimulated oocytes of Pisaster ochraceus also undergo a decrease in cAMP content, the size of which is increased by forskolin. Although a decrease in cAMP begins at sub-threshold 1-MeAde concentrations, the maximal decrease occurs at the same concentration of 1-MeAde needed for maturation induction and a further 1000-fold increase of the 1-MeAde concentration has no further effect. Upon removal of 1-MeAde, the cAMP concentration immediately increases to its original level. Sequential addition and removal of 1-MeAde triggers a sequential decrease and increase of the cAMP concentration, illustrating the continuous requirement for 1-MeAde for eliciting the decrease. Successive additions of 1-MeAde, however, do not trigger further decreases of the cAMP concentration. The temperature dependences of the cAMP concentration decrease and of the hormone-dependent period (HDP; the time of contact with 1-MeAde required for induction of maturation) are closely related. Forskolin, which increases the cAMP concentration, also increases the duration of the HDP (2.5-fold), delays the time course of protein phosphorylation burst and germinal vesicle breakdown, and inhibits AA- and 8-HETE-induced maturation. We conclude that 1-MeAde triggers a drop in cAMP concentration, which is tightly associated with the hormone-dependent period of oocyte maturation.     

INDUCTION OF OOCYTE MATURATION BY DISULFIDE-REDUCING AGENT IN THE SEA CUCUMBER, STICHOPUS JAPONICUS

1-Methyladenine (1-MeAde) is known to be a natural inducer of starfish oocyte maturation. Disulfide-reducing agents such as dithiothreitol (DTT) and 2, 3-dimercapto-1-propanol (BAL) are known to mimic the action of 1-MeAde in inducing starfish oocyte maturation. Although 1-MeAde failed to induce oocyte maturation in sea cucumbers, breakdown of germinal vesicles and subsequent meiotic behaviour of chromosomes were induced by the treatment with DTT in the pronase-treated oocytes of the sea cucumber, Stichopus japonicus. These findings suggest that reduction of disulfide bonds plays an important role in triggering oocyte maturation in some marine forms such as echinoderms.     

EFFECT OF LOCAL APPLICATION OF 1-METHYLADENINE ON THE SITE OF POLAR BODY FORMATION IN STARFISH OOCYTE*

Mechanism by which the site of polar body formation is determined in starfish oocytes was investigated in relation to the action of 1-methyladenine (1-MeAde). Local staining with Nile Blue of Asterina pectinifera oocytes revealed that there exists a prospective site of polar body formation (PSPBF) on the nearest surface to the position of germinal vesicle. The site of polar body formation was found to shift to some extent from PSPBF toward the area locally applied with 1-MeAde, suggesting that the actual site of polar body formation is not determined yet at the germinal vesicle stage. Oocytes whose germinal vesicles had been shifted by centrifugation from PSPBF to the opposite surface before the commencement of germinal vesicle breakdown (GVBD) (less than 15 min after 1-MeAde treatment), failed to form polar bodies, whereas oocytes centrifuged after commencement of GVBD (20 min after 1-MeAde treatment) did form polar bodies where their fading germinal vesicles had reached by centrifugation. In the oocytes which failed to form polar bodies by centrifugation, an aster was observed near PSPBF of each oocyte. When inseminated, every oocyte treated with 1-MeAde developed normally irrespectively of the mode of polar body formation including the site and the occurrence, and the animal pole of every larva was derived from PSPBF.     

Inhibition of starfish oocyte maturation by some inhibitors of proteolytic enzymes

Starfish oocyte maturation is triggered by a natural hormone, 1-methyladenine (1-MeAde), produced in the follicle cells, or artificially by dithiothreitol (DTT). These substances act on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which induces germinal vesicle breakdown (GVBD) and subsequent processes of meiotic maturation. Further, MPF is amplified in immature oocytes that have received the injection of MPF. In this paper the effect of leupeptin and antipain, protease inhibitors of microbial origin, on starfish oocyte maturation was investigated. The protease inhibitors were found to inhibit 1-MeAde-induced maturation when they were applied externally or injected into oocytes. DTT-induced maturation was also inhibited by injection of leupeptin. However, leupeptin did not inhibit the maturation-inducing action of MPF or MPF amplification. These results show that the protease inhibitors suppress the production of MPF by 1-MeAde or DTT, suggesting that some endogenous protease(s) acts in the production of MPF.     

MEK1/2 is a critical regulator of microtubule assembly and spindle organization during rat oocyte meiotic maturation

MEK (MAPK kinase) is an upstream protein kinase of MAPK in the MOS/MEK/MAPK/p90rsk signaling pathway. We previously reported the function and regulation of MAPK during rat oocyte maturation. In this study, we further investigated the localization and possible roles of MEK1/2. First, immunofluorescent staining revealed that p-MEK1/2 was restricted to the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), p-MEK1/2 condensed in the vicinity of chromosomes and then translocated to the spindle poles at metaphase I, while spindle microtubules stained faintly. When the oocyte went through anaphase I and telophase I, p-MEK1/2 disappeared from spindle poles and became associated with the midbody. By metaphase II, p-MEK1/2 was again localized to the spindle poles. Second, p-MEK1/2 was localized to the centers of cytoplasmic microtubule asters induced by taxol. Third, p-MEK1/2 co-localized with gamma-tubulin in microtubule-organizing centers (MTOCs). Forth, treatment with U0126, a non-competitive MEK1/2 inhibitor, did not affect germinal vesicle breakdown, but caused chromosome mis-alignment in all MI oocytes examined and abnormal spindle organization as well as small cytoplasmic spindle-like structure formation in MII oocytes. Finally, U0126 reduced the number of cytoplasmic asters induced by taxol. Our data suggest that MEK1/2 has regulatory functions in microtubule assembly and spindle organization during rat oocyte meiotic maturation.     

Oocyte-surface factor responsible for 1-methyladenine-lnduced oocyte maturation in starfish

Fully grown oocytes of the starfish Asterina pectinifera, undergo breakdown of their germinal vesicles and subsequent maturation on treatment with 1-methyladenine (1-MeAde). However, oocytes treated with seawater containing 0.010% Triton X-100 lost the capacity to respond to 1-MeAde and their germinal vesicles remained intact. These decapacitated oocytes once again ac-quired the capacity to respond to l-Me Ade when they were incubated in sea water containing the extract of fully grown oocytes treated with Triton X-100, from which the Triton X-100 was removed after extraction by means of Bio-Beads SM-2 (TXE). Recovery of the capacity was also observed after washing such TXE-treated oocytes with sea water. These results suggest that some factor (probably 1-MeAde receptor or its fragment), extracted from the oocyte surface (plasma mem-brane) by nonionic detergent, was reconstituted on the oocyte surface so that the capacity of the oocytes to respond to 1-MeAde was recovered. The factor was heat-stable and resistant to treat-ment with proteolytic enzymes.     

Activation of maturation promoting factor and 26S proteasome assembly accelerated by a high concentration of 1-methyladenine in starfish oocytes

In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-norvalinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation-inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation.     

MARKED DECREASE IN THE RIGIDITY OF STARFISH OOCYTES INDUCED BY 1-METHYLADENINE1

Extreme rigidity of immature starfish oocytes as measured by compression method was found to decline during the early phase of their maturation when induced by 1-methyladenine (1-MeAde). The onset of this decrease in stiffness occurred within 5 to 9 min of 1-MeAde treatment, well before the breakdown of the germinal vesicle, progressively declining to reach a minimum stiffness after 20 min. Dithiothreitol, known as an artificial maturation-inducing agent, caused a similar change. The stiffness is thus expected to serve as a quantitative indicator of the early process of cytoplasmic events, which would induce the breakdown of the germinal vesicle. Cytochalasin B (3 μg/ml) also reduced the stiffness, but unlike the former two agents, the effect was reversible, and did not interfere with the process of maturation. Due to the effect of cytochalasin B, it became possible to enucleate immature oocytes by centrifugal force. Non-nucleate fragments thus obtained still maintained their marked stiffness, which was decreased by the action of 1-MeAde, with a time-course similar to that of intact oocytes.     

Breakdown of starfish ovarian follicle induced by maturation-promoting factor

Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.     

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.     

Cytoplasmic Maturity Revealed by the Structural Changes in Incorporated Spermatozoon during the Course of Starfish Oocyte Maturation

Immature oocytes of the starfish, Asterina pectinifera, are polyspermic. Spermatozoa can enter immature oocytes upon insemination, but the changes associated with the fertilization process in oocytes matured with 1-methyladenine (1-MeAde), such as the formation of aster and pronucleus, were not observed. After immature oocytes, previously inseminated, were matured with 1-MeAde, the formation of the sperm monaster was observed during germinal vesicle breakdown (GVBD). Amphiasters and pronuclei were formed after the formation of the second polar body. The acquisition by oocytes of the capacity to undergo the normal process of fertilization, therefore, occurs during the course of oocyte maturation. After injection of the cytoplasm of maturing oocytes into inseminated immature oocytes, the formation of aster and pronucleus was observed, suggesting that maturation-promoting factor (MPF) may be involved in establishing the cytoplasmic conditions (cytoplasmic maturity) necessary for the fertilization process to occur. In contrast, when enucleated, inseminated halves of immature oocytes were treated with 1-MeAde, only monasters were formed, while in the nucleated halves both amphiasters and sperm pronuclei were formed. Thus, germinal vesicle material is required for the formation of amphiaster and sperm pronucleus but not for the formation of monaster. It is possible that the amount of MPF produced in enucleated halves was sufficient only for the formation of the monaster but not for the formation of the amphiaster and pronucleus, since it has been previously established that germinal vesicle material is necessary for the amplification of MPF. The formation of the monaster in the enucleated halves at a time corresponding to GVBD in nucleated controls suggests that the amount of MPF needed for this event is rather small. For the induction of subsequent fertilization process, large amounts of MPF may be required to establish the necessary cytoplasmic conditions, although other possible role of nuclear material is not excluded.     

Assembly of a protein sharing epitopes with sperm dynein heavy chains into meiotic spindle in the prometaphase starfish oocyte

Starfish oocyte meiosis provides a good system for studying the mechanism for prometaphase chromosome movement. Since a protein sharing epitopes with sperm dynein might be a force generator for mitosis, the contribution of such a protein was assessed in this movement. Specific antibodies to heavy chains (HCs) and intermediate chains (ICs) of dynein subunits were affinity-purified from whole antidynein serum. We confirmed that the oocytes contain several polypeptides identical to sperm dynein subunits. The anti-HCs binding to in situ antigen was examined in the oocytes permeabilized with detergent at appropriate stages of maturation with special reference to tubulin and chromosomes, and the meiotic apparatus-establishing process was described in terms of a force generator (oocyte dynein). Before resumption of maturation, dynein HCs were particularly associated with prophase chromosomes within the germinal vesicle (GV). After GV breakdown, there was a striking local accumulation of dynein HCs in the "fading GV" (nuclear matrix). When chromosomes were pulled toward the central area between 2 asters, dynein was accumulated at first at the presumptive equator and then moved to the poles, showing uneven localization on the meiotic spindle.     

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.