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.     

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.     

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.     

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.     

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.     

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.     

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.     

Transforming growth factor-alpha augments meiotic maturation of cumulus cell-enclosed mouse oocytes

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor-alpha (TGF-alpha) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell-enclosed oocytes were exposed to TGF-alpha with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denuded oocytes were examined for GVBD following exposure to HX and TGF-alpha. When cumulus cell-enclosed oocytes were exposed to TGF-alpha (1 microgram/ml) in the presence of HX (4 mM), an increase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF-alpha and HX as compared to 33% with HX only. Concentrations of TGF-alpha as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF-alpha, but without HX, was also enhanced. The stimulation of GVBD by TGF-alpha showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF-alpha in the presence of HX, no effect was observed. Our results suggest that TGF-alpha is a potent stimulator of mouse oocyte maturation in vitro and that its effect is mediated by the surrounding cumulus cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

One Millimeter large Oocytes as a Tool to Study Hormonal Control of Meiotic Maturation in Starfish: Role of the Nucleus in Hormone-stimulated Phosphorylation of Cytoplasmic Proteins

Single nuclei (germinal vesicles) manually isolated from large oocytes of the starfish Echinaster sepositus , as well as the complementary anucleated oocytes, were used to investigate the early changes of protein phosphorylation which occur from 1-MeAde addition to germinal vesicle breakdown (GVBD). Stimulation of protein phosphorylation was already evident in the nucleus shortly after 1-MeAde addition (18 min, thus about 0.40x the time required for GVBD), although it began first in the cytoplasm. No translocation of phosphoprotein across the nuclear envelope was detected before GVBD. Presence of the nucleus is not required for the hormone to stimulate protein phosphorylation in the remaining part of the oocytetin:fact the patterns of protein phosphorylation in enucleated oocytes were found to be identical, whether enucleation was performed after or before hormonal treatment. Cytoplasm taken at the time of GVBD from maturing Echinaster oocytes induces meiotic maturation when transferred in stage VI immature oocytes of the amphibian Xenopus laevis.     

Dynamics of Mitotic Apparatus Formation and Tubulin Content during Oocyte Maturation in Starfish

To follow the topo-temporal behavior of structures containing tubulin and the change in tubulin content during oocyte maturation, starfish oocytes were extracted with a medium containing detergent so that morphological observation and biochemical analysis could be conducted on the same residual oocyte preparation simultaneously. Before 1-methyladenine (1-MeAde) stimulation, "pre-meiotic asters" were observed on the germinal vesicle at the animal pole. 1-MeAde caused the appearance of distinct asters at the position of the aster precursor. When germinal vesicle breakdown (GVBD) took place, chromosomes were condensed. Chromosome gathering was concurrent with a reduction in the size of nuclear matrix. The mitotic apparatus was first constructed parallel to the cortex and then changed its axis perpendicularly. Fluorescence of tubulin due to indirect immunofluorescence in the cytoplasm other than the mitotic apparatus decreased rapidly along the course of maturation at least up to the first metaphase. Despite these dynamic morphological change, the tubulin content in the whole oocyte and the residual structures, measured by SDS-PAGE and immunostaining, did not show remarkable (statistically significant) changes through the course of maturation, although the content tended to decrease a little before the second polar body formation and to increase thereafter in the latter.     

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.     

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.     

Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin

The efficacy of follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)-containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP-supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte-cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP-dependent process. The frequency of FSH- and EGF-induced GVBD in cumulus cell-enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. of maturation is apparently not mediated solely by oocyte-cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.     

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.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish

Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.     

Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes

In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.     

Nature of the 1-Methyladenine-Requiring Phase in Maturation of Starfish Oocytes

Reinitiation of meiosis in starfish oocytes requires the continuous presence of 1-methyladenine (1-MeAde) in the surrounding medium for a definite period. The length of the 'hormone-dependent phase' (HDP) in Asterina pectinifera , which was defined as the time necessary for induction of 50% germinal vesicle breakdown (GVBD), was found to be about 11 min at 17°C, and 8 min at 20°C. Repeated treatments for shorter periods with 1-MeAde revealed that the action of this agent was cumulative, and that stable intermediate states between the unstimulated and fully stimulated levels existed during the HDP. Measurement of the stiffness of oocytes also demonstrated this stable intermediate state. Thus, there may be a factor(s) in the cytoplasm that accumulates continuously during the HDP and triggers GVBD when it reaches a critical level(s). When dithiothreitol (DTT) was used as an artificial maturation-inducing agent, the intermediate state was far less stable, suggesting a difference in the modes of action of 1-MeAde and DTT. Isotonic CaCl₂, the Ca²⁺ ionophore (A 23187) and methylxanthines, which are known to cause increase in intracellular Ca²⁺, had additive effects with 1-MeAde. These results suggest that part of the action of 1-MeAde is to release Ca²⁺ in the oocyte cytoplasm.