Polar Body Emission Research Articles

Pronuclear Migration and Cytoskeletal Organization of Porcine Oocytes Activated by Various Stimuli

A variety of physical and chemical stimuli initiate egg activation events. This study was designed to investigate the rates of pronuclear formation and the cytoskeletal organization of porcine oocytes activated by various stimuli. Oocytes matured for 44-60 hrs were activated by one of the following treatments: double electric pulses (EP, 150 V/mm for 60 µsec, 1 sec apart), ethanol (7% for 5 min), calcium ionophore A23187 (CaA, 50 µM for 2 min), cycloheximide (CHX, 5 µg/ml for 6 hrs), or 6-dimethylaminopurine (DMAP, 2.5 mM for 4 hrs). EP yielded the highest activation rate (97%). CaA and DMAP activated the oocytes in relatively higher proportions than did ethanol and CHX (74 and 83% vs. 55 and 41%, respectively). After CHX and DMAP treatments, egg fragmentation was significantly increased (65 and 82%, respectively) and the polar body emission was restrained. After activation with EP, ethanol or CaA, the pronucleated oocytes possessed a microtubule-rich domain around the pronucleus, which was concomitantly surrounded by microfilaments. In oocytes activated with CHX or DMAP, however, microtubules and microfilaments did not synchronously form a concentrated domain. We suggest that activation with a protein synthesis/phosphorylation inhibitor may have affects on distribution and function of the cytoskeleton of the oocyte different from those elicited by treatments with EP, ethanol or CaA, which result in higher proportions of eggs with the pronucleus restrained in the peripheral ooplasm and greater egg fragmentation.

Expression pattern of HSP25 in mouse preimplantation embryo: heat shock responses during oocyte maturation.

Heat shock proteins (HSPs) are known to play an important role not only in various stress conditions such as exposure to heat shock, but also in normal development and/or differentiation. The role of small heat shock proteins such as HSP25 in early embryo development remains largely unknown. In the present study, we examined temporal and spatial expression patterns of HSP25 during mouse preimplantation embryo development. Reverse transcription-polymerase chain reaction (RT-PCR) showed that hsp25 mRNA was detected in unfertilized eggs. Hsp25 mRNA was induced by zygotic gene activation at 2-cell stage, decreased slightly at 4-cell, and re-increased at morula, with the highest level at blastocyst stage. Interestingly, another form of hsp25 variant of which 156 bp (52 a.a.) was truncated within the exon1 region was observed in all stages of preimplantation embryos. We also investigated the sub-cellular localization of HSP25 by fluorescence immunocytochemistry. HSP25 was detected in the cytoplasm under normal developmental condition. While acute heat shock (at 43 degrees C for 30 min) caused no significant changes in the sub-cellular localization of HSP25 in the developing mouse embryos, chronic heat shock (at 43 degrees C for 3 hr) resulted in a denser immunostaining of HSP25 in the nucleus than in the cytoplasm, indicating a nuclear translocation of HSP25 by heat shock. As hsp25 mRNA was detected in the unfertilized egg as a maternal transcript, we examined the expression of hsp25 mRNA with RT-PCR during oocyte maturation under normal and heat shock conditions. Hsp25 mRNA was detected at GV (germinal vesicle)-, GVBD (germinal vesicle breakdown)-, and MII (metaphase II)-oocytes. The expression of hsp25 mRNA was increased markedly by both acute (for 30 min and 1 hr) and chronic (for 4 hr) heat shock, but returned to the basal level during recovery from heat shock in a time-dependent manner, suggesting a thermo-protective role of HSP25. In contrast to preimplantation embryos, HSP25 was detected both in the cytoplasm and the nucleus except for the nucleolus, and the cellular localization was not altered by heat shock. Finally, we investigated the effect of heat shock on oocyte maturation. When GV-oocytes were exposed to acute heat shock (at 43 degrees C for 15 min to 1 hr), they underwent the GVBD and the PB (polar body) emission successfully. However, under more stringent heat shock conditions (at 43 degrees C for 2-4 hr), most oocytes were arrested at the GV-stage, and the first PB was not developed, indicating that chronic heat shock might be inhibitory to the mouse oocyte maturation. Taken together, these findings suggest that HSP25 is important for mouse preimplantation embryo development and oocyte maturation.

Fertilization Blocks Apoptosis of Starfish Eggs by Inactivation of the MAP Kinase Pathway

Fully grown starfish oocytes are arrested at prophase of meiosis I. The hormonal stimulation of 1-methyladenine (1-MA) induces meiosis reinitiation and germinal vesicle breakdown (GVBD). Optimal development occurs when maturing oocytes are fertilized between GVBD and first polar body emission. In the absence of sperm, oocytes complete both meiotic divisions to yield haploid interphase-arrested eggs. We now report that spontaneous and synchronous activation of caspase-3 in starfish eggs occurs 9–12 h after 1-MA stimulation. Then, caspase-dependent membrane blebbing and egg fragmentation occur, indicating that mature eggs undergo apoptosis if not fertilized. Activation of caspase-3 and induction of apoptosis are blocked both by a MEK inhibitor and by emetine treatment which inhibits MEK kinase (Mos) synthesis. Conversely, when recombinant GST-Mos is injected into the emetine-treated eggs, apoptosis is induced. These results indicate that persistent activation of the Mos/MEK/MAP kinase cascade gives the death-activating signal in starfish eggs. Fertilization inactivates the MAP kinase pathway and suppresses apoptosis, followed by normal development.

Mouse‐rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species‐specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0–14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage. J. Exp. Zool. 289:322–329, 2001. © 2001 Wiley-Liss, Inc.

Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes.

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI. Mol. Reprod. Dev. 56:51–62, 2000. © 2000 Wiley-Liss, Inc.

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes.

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.

Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice.

Jasplakinolide (JAS), which induces microfilament polymerization and stabilization, inhibits microfilament-mediated events in murine oocyte maturation and fertilization in a fashion unlike the effects of cytochalasin B (CCB) and latranculin A (LAT A). JAS prevents egg polar body emission at a much lower concentration than either CCB or LAT A. Microfilament bundles were detected on the entire egg cortex after JAS exposure. Conversely, microfilament patterns did not change after exposure to CCB, and few microfilaments were observed after exposure to LAT A. Eggs that were allowed to recover from JAS were unable to recover normal microfilament organization. During oocyte maturation, JAS prevented both spindle migration to the oocyte cortex and first polar body emission. During in vitro fertilization, sperm head entered the eggs and formed pronuclei, but sperm tail entry, pronuclear centration, and second polar body emission were not detected. DNA synthesis occurs in these JAS-treated zygotes. JAS inhibited not only the formation, but also the disassembly, of incorporation cones. JAS was also found to prevent cortical granule exocytosis following artificial activation, and cortical granules were still beneath the plasma membrane even after activation. Finally, incorporation of microinjected nonmuscle actin into the microfilament network of mice eggs was delayed by JAS. We conclude that JAS acts as a microfilament inhibitor during maturation and fertilization and is more powerful than other inhibitors. Its mechanism differs in that it promotes assembly and stabilization of microfilaments. JAS is a novel cell permeable tool for the investigation of microfilament-dependent events in early mammalian development.

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes.

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.

Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes,Without influencing chromosome condensation activity.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

Mitogen-activated protein kinase and cell cycle progression during mouse egg activation induced by various stimuli.

A very sensitive method was established for detecting the activity of mitogen-activated protein (MAP) kinase in mouse eggs, and used to follow temporal changes of this kinase during fertilization and spontaneous or chemically-induced parthenogenic activation. MAP kinase activity increased between 1 and 2.5 h post-insemination, at which time the second polar body was emitted and sperm chromatin was dispersed; its activity decreased sharply at 8 h. when pronuclei were formed. Both calcium ionophore A23187 and ethanol simultaneously induced pronuclear formation and MAP kinase inactivation in aged eggs 8 h after incubation but less effectively in fresh eggs. The protein kinase inhibitor staurosporine induced pronuclear formation and MAP kinase inactivation more quickly than other treatments, with MAP kinase inactivation occurring slightly proceeding pronuclear formation. Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, induced increase in MAP kinase activity, and overcame pronuclear formation induced by various stimuli. MAP kinase inactivation preceded pronuclear formation in eggs spontaneously activated by aging in vitro, perhaps due to cytoplasmic degeneration and thus delayed response of nuclear envelope precursors to MAP kinase inactivation. These data suggest that MAP kinase is a key protein kinase regulating the events of mouse egg activation. Increased MAP kinase activity is temporally correlated with the second polar body emission and sperm chromatin decondensation. Although different stimuli (including sperm) may initially act through different mechanisms, they finally inactivate MAP kinase, probably by allowing the action of protein phosphatase, and thus induces the transition to interphase.

Tyrosine phosphorylation of p34cdc2 in metaphase II-arrested pig oocytes results in pronucleus formation without chromosome segregation

At the G2-M boundary, maturation-promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34cdc2 or synthesis of cyclin B according to cell type and species. At the end of M-phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34cdc2 can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)-arrested oocytes. First, cyclin B1 in MII-arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34cdc2 rather than cyclin B1 degradation. The vanadate-induced exit from M-phase is distinct from normal M-phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin-dependent kinases, they exited from MII arrest in a manner similar to vanadate-treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34cdc2 enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase-promoting complex (APC)-mediated proteolysis, on the signaling pathway from intracellular Ca2+ increase to MPF inactivation. Mol. Reprod. Dev. 52:107–116, 1999. © 1999 Wiley-Liss, Inc.

Tyrosine phosphorylation of p34cdc2 in metaphase II‐arrested pig oocytes results in pronucleus formation without chromosome segregation

At the G2-M boundary, maturation-promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34cdc2 or synthesis of cyclin B according to cell type and species. At the end of M-phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34cdc2 can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)-arrested oocytes. First, cyclin B1 in MII-arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34cdc2 rather than cyclin B1 degradation. The vanadate-induced exit from M-phase is distinct from normal M-phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin-dependent kinases, they exited from MII arrest in a manner similar to vanadate-treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34cdc2 enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase-promoting complex (APC)-mediated proteolysis, on the signaling pathway from intracellular Ca2+ increase to MPF inactivation. Mol. Reprod. Dev. 52:107–116, 1999. © 1999 Wiley-Liss, Inc.

Biochemical and biological effects of KN-93, an inhibitor of calmodulin-dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol.

Calmodulin-dependent protein kinase II (CaMKII) is transiently activated in mouse eggs by the increase in calcium that occurs upon activation with ethanol. This study investigated the biological and biochemical effects of KN-93, a reported selective inhibitor of CaMKII, to explore the potential role of this kinase in the initial events of egg activation. Mouse eggs were incubated for 30 min in the presence of different concentrations of KN-93 and induced to activate by 7% ethanol. KN-93 elicited a dose-dependent inhibition of polar body emission that resulted from the failure of the eggs to undergo meiosis resumption and inactivation of maturation-promoting factor (MPF). Furthermore, 15 mumol KN-93 l-1 produced a marked reduction in ethanol-induced loss of cortical granules. In vivo biochemical analysis revealed that 15 mumol KN-93 l-1 was responsible for significant inhibition of ethanol-stimulated CaMKII. The activity of the enzyme remained at a resting value, in spite of the presence of a calcium signal similar to that measured in control activated eggs. The inhibitory effects of KN-93 on the parameters tested in this study could not be mimicked by the inactive analogue KN-92. These results show that in mouse eggs, when ethanol-induced CaMKII activation was prevented, cortical granule exocytosis and meiosis resumption were inhibited. This suggests that CaMKII acts as a switch in the transduction of the calcium signal triggering mammalian egg activation.

Role of Two Series of Ca2+Oscillations in Activation of Ascidian Eggs

Changes in [Ca2+]iare an essential factor regulating egg activation. Matured ascidian eggs are arrested at metaphase I, and two series of [Ca2+]itransients have been observed after fertilization: Ca2+waves just after fertilization (Series I) and [Ca2+]ioscillation between the first and second polar body extrusion (Series II). We investigated mechanisms involved in the elevation of [Ca2+]iand the role of the [Ca2+]itransients during egg activation inCiona savignyi.The monoclonal antibody 18A10 against IP3receptor type 1, which inhibits IP3-induced Ca2+release in hamster and mouse eggs, did not show substantial inhibitory effects on series I or egg deformation, whereas Series II and the first cell division were inhibited by the antibody. Ruthenium red, an inhibitor of ryanodine receptor-mediated Ca2+release, had no apparent effect of [Ca2+]itransients and other events related to the egg activation. Microinjection of IP3into unfertilized eggs induced [Ca2+]itransients similar to those seen in Series I, whereas injection of cyclic ADP ribose, an agonist of ryanodine receptors, rarely induced [Ca2+]itransient. Adenophostin B, a potent nonmetabolizable agonist of IP3receptors, induced [Ca2+]ioscillations which continued after first polar body extrusion, without separation to two series, and led to extrusion of first and second polar bodies. These results suggest that Series II is driven by the mouse type 1-like IP3receptor while Series I seems to be mediated by another type of IP3receptor. Injection of IP3only induced the first polar body extrusion and the egg was arrested at metaphase II even when a higher amount of IP3was injected. On the other hand, reinjection of IP3after the first polar body extrusion led to emission of the second polar body. Thus, Series I and II of [Ca2+]itransients are likely to be required for metaphase–anaphase transition in meiosis.

Protein kinase inhibitors induce the interphase transition by inactivating mitogen-activated protein kinase in mouse eggs.

The role of mitogen-activated protein (MAP) kinase in mouse egg activation induced by protein kinase inhibitors and a protein tyrosine kinase (PTK) inhibitor was investigated. Separated egg proteins were first probed with anti-Active MAP kinase antibody and then re-probed with anti-ERK2 antibody. Staurosporine and Ro-31-8220, at concentrations that normally inhibit protein kinase C, did not affect egg activation or MAP kinase activity, while higher dosages caused egg activation. Staurosporine at 2 microM induced the metaphase-interphase transition without emission of the second polar body (PB2), while Ro-31-8220 at 40 microM induced PB2 emission, first cleavage, and then the transition to interphase. Half the eggs were also activated by the PTK inhibitor genistein. In each treatment, the proportion of eggs that entered interphase was well correlated with the degree of MAP kinase inactivation. Artificial activation of this kinase by okadaic acid overcame the interphase transition. These data suggest that protein kinase inhibitors and a protein tyrosine kinase inhibitor induce the interphase transition by inactivating MAP kinase in mouse eggs.

Changes in Cyclin B during Oocyte Maturation and Early Embryonic Cell Cycle in the Newt,Cynops pyrrhogaster: Requirement of Germinal Vesicle for MPF Activation

When full-grown oocytes of the newtCynops pyrrhogasterwere treated with progesterone in O-R2 solution containing antibiotics, approximately 85% of the oocytes completed meiosis synchronously. Maturation-promoting factor (MPF) activity appeared just before germinal vesicle breakdown (GVBD) and the oocytes maintained high MPF activity throughout metaphase I and metaphase II of meiosis. A slight decrease of MPF activity was observed at the first polar body emission. The distribution of cyclin B1 was investigated with anti-cyclin B1 antibody. No cyclin B1 was found in the oocytes before progesterone treatment. Cyclin B1 appeared in the cortex of animal hemispheres, especially around and inside germinal vesicle just before GVBD. A large amount of cyclin B1 accumulated at metaphase I, approximately half disappeared at the first polar body emission, and then cyclin B1 accumulated again at metaphase II. An inactive form of cdc2 kinase was observed in both the germinal vesicles and the oocyte cytoplasm, while an active form appeared at the M phase. No MPF was observed in the oocytes from which the germinal vesicle had been removed. A cdk7-like molecule was localized in the germinal vesicle, but not in oocyte cytoplasm, indicating that inactive cdc2 kinase associated with cyclin B1 derived from cytoplasm is activated by phosphorylation in the germinal vesicle. The changes in the amount of cyclin B1 were synchronous with the first cell cycle after fertilization. Cyclin B1 was primarily localized in the cortex of the animal hemisphere. A shift in band mobility upon electrophoresis of cyclin B1 was observed from samples taken during the cell cycle; this shift was probably due to the protein's phosphorylation state.

The Two Intracellular Ca2+Release Channels, Ryanodine Receptor and Inositol 1,4,5-Trisphosphate Receptor, Play Different Roles during Fertilization in Ascidians

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs ofPhallusia mammillatausing whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3(10 μMperfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15–20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.

Separation of Female Chromosomes is Specifically Induced by Protein Kinase C Independent of the Calcium Pathway during Fertilization of Mouse Eggs.

To evaluate the function of protein kinase C (PKC) during oocyte activation following fertilization, the effects of activators and inhibitors of PKC on the second polar body emission, pronuclear formation and protein phosphorylation of mouse oocytes were investigated during in vitro fertilization and during artificial oocyte activation with Ca ionophore (A23187). One ng/ml of 12-O-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, accelerated PF without accelerating the second polar body emission. 5-20 ng/ml of TPA suppressed the second polar body emission significantly but did not inhibit female chromosome separation of second meiotic division. Staurosporine, a PKC inhibitor, at 1-5 nM, suppressed the second polar body emission. Female chromosome separation was also suppressed by staurosporine and H7, an another PKC inhibitor. One diploid female pronucleus was formed in an egg or an oocyte without the second polar body emission. TPA itself caused female chromosome separation even in the presence of 20 μM of BAPTA-AM, a selective membrane-permeable calcium chelator. These results show that PKC has supportive effect on pronuclear formation. The activation of PKC is indispensable for female chromosome separation during fertilization.