Actin depolymerization is associated with meiotic acceleration in cycloheximide-treated ovine oocytes.

Oocytes treated with the protein synthesis inhibitor cycloheximide (CHX) arrest at the germinal vesicle (GV) stage and undergo accelerated GV breakdown (GVBD) after CHX is removed. However, little is known about the underlying mechanism of accelerated meiotic maturation. Here, we investigated this mechanism and found that oocytes released from CHX arrest have higher amounts of cyclin B1 (CCNB1) and phosphorylated mitogen-activated protein kinase (pMAPK) proteins. Increased levels of these factors were not associated with mRNA polyadenylation or increased transcription rates of CCNB1 and MOS (Moloney murine sarcoma viral oncogene homolog) during CHX arrest. We found that treatment of CHX-arrested oocytes with the actin filament-stabilizing agent Jasplakinolide (Jasp) delayed GVBD following release from CHX arrest and that this was correlated with reduced maturation-promoting factor (MPF) activity. These results suggest that CCNB1 mRNAs released from actin filaments during CHX arrest increase CCNB1 transcripts available for translation after release from CHX arrest, leading to the precocious activation of MPF and accelerated meiotic progression.

A primer on meiotic resumption in starfish oocytes: the proposed signaling pathway triggered by maturation-inducing hormone.

This short review updates the maturation-inducing hormonal signaling in starfish oocytes. In this system, the activation of cyclin B-Cdc2 kinase (Cdk1) that leads to meiotic resumption does not require new protein synthesis. The key intracellular mediator after hormonal stimulation by 1-methyladenine is the protein kinase Akt/PKB, which in turn directly downregulates Myt1 and upregulates Cdc25 toward the activation of cyclin B-Cdc2. Mitotic kinases including Aurora, Plk1 and Greatwall are activated downstream of cyclin B-Cdc2. The starfish oocyte thus provides a simple model system for the study of meiotic resumption.

Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla.

The influences of serotonin (5-hydroxytryptamine) on the action of melatonin (N-acetyl-5-methoxytryptamine) in MIH (maturation inducing hormone)-induced meiotic resumption were evaluated in the oocytes of carp Catla catla using an in vitro model. Oocytes from gravid female carp were isolated and incubated separately in Medium 199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 microg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 microM) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 microM) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. Control oocytes were incubated in the medium alone. Oocytes were incubated for 4, or 8, or 12, or 16 h and effects were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). At the end of 16 h incubation, 93.24+/-1.57% oocytes underwent GVBD following incubation with only MIH, while incubation with only MEL or only SER resulted in 77.15+/-1.91% or 14.42+/-0.43% GVBD respectively. Interestingly, incubation with MEL 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD (92.58+/-1.10% at 12 h). In contrast, SER, irrespective of its time of application in relation to MIH, resulted in a maximum of 64.57+/-0.86% GVBD. While L was found to reduce the stimulatory actions of melatonin, M suppressed the inhibitory actions of serotonin. In each case, both electrophoretic and immunoblot studies revealed that the rate of GVBD was associated with the rate of formation of maturation promoting factor (a complex of two proteins: a regulatory component--cyclin B and the catalytic component--Cdk1 or cdc2). Collectively, the present study reports for the first time that SER not only inhibits the independent actions of MIH, but also the actions of MEL on the MIH-induced oocytes maturation in carp.

Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer.

When the nucleus in G0/G1 phase is transferred to an enucleated oocyte by nuclear transfer (NT), its nuclear envelope is broken, followed by condensation of chromosome structure by maturation promoting factor (MPF). This morphological remodeling of the transferred interphase nucleus seems to be essential for subsequent development of NT embryos. In this study, we treated porcine NT embryos with caffeine, which has been reported to increase MPF activity, to keep their MPF level high during NT. When 2.5 mM caffeine was added to the handling medium, the proportion of NT embryos showing condensed chromosome increased significantly (P < 0.05). In NT embryos treated with caffeine, the activity of p34(cdc2) kinase was significantly (P < 0.05) higher than in those without caffeine at 3 h post-injection. In addition, the rate of development to the blastocyst stage after activation was significantly (P < 0.05) higher in NT embryos treated with caffeine. These results indicate that caffeine treatment can increase not only the rate of chromosome condensation but also the developmental rate to the blastocyst stage of porcine NT embryos. This action is most likely due to the support/increase of MPF activity throughout the process of NT.

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes.

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.

Endocrine control of diurnal oocyte maturation in the kyusen wrasse, Halichoeres poecilopterus.

The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20beta-P and 20beta-S increased around the time of GVBD and ovulation, suggesting that 17,20beta-P and 20beta-S act as MIHs in the kyusen wrasse. Moreover, in vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.

Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts.

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.

Rescue of mouse embryos from 2-cell blocks by microinjection of maturation promoting factor.

OBJECTIVE: To examine the rescue of mouse embryos from 2-cell blocks by the microinjection of maturation promoting factor (MPF) extracted from matured Xenopus eggs into one of the blastomeres of 2-cell stage mouse embryos. DESIGN: Controlled laboratory study. SETTING: First Department of Obstetrics and Gynecology, Toho University School of Medicine, Tokyo, Japan. ANIMAL(S): Eight- to 10-week-old female Crj:CD-1(ICR) mice. INTERVENTION(S): One of the blastomeres of the mouse 2-cell embryos was injected with MPF (MI group) or mHTF medium (MED group) at 28--32 hours after insemination. MAIN OUTCOME MEASURE(S): The developmental rate to blastocyst. RESULT(S): The developmental rate to blastocyst in the MI group (48.0%) was significantly higher than that in the MED group (0%). CONCLUSION(S): The 2-cell block was specifically rescued by the microinjection of MPF and not by the insertion of pipettes.

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.

Study of the in vitro maturation of mouse oocytes induced by microinjection of maturation promoting factor (MPF).

PURPOSE: Maturation promoting factor (MPF) acts at the resumption of meiosis and nonspecifically throughout the animal species. There exists a considerable body of literature on MPF, but little work has been done to study the induction of maturation of mammalian oocytes by microinjection of extracted MPF. METHODS: Immature (GV-stage) mouse oocytes were microinjected MPF extracted from matured Xenopus eggs in the presence of dbcAMP. RESULTS: The rate of germinal vesicle, breakdown (GVBD) induced at 24 hr after MPF injection was significantly higher (90.5%) than that of the control (2.2%), which was injected with HTF medium containing dbcAMP (P < 0.0001). The rate of extrusion of the first polar body at 24 hr after MPF injection was significantly higher (84.1%) than that of the same control (1.1%) (P < 0.0001). CONCLUSIONS: From these results, it is concluded that the maturation of mammalian oocytes can be induced by the microinjection of MPF extracted from other species.

A p90(rsk) mutant constitutively interacting with MAP kinase uncouples MAP kinase from p34(cdc2)/cyclin B activation in Xenopus oocytes.

The efficient activation of p90(rsk) by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90(rsk). The MAP kinase p42(mpk1) can associate with p90(rsk) in G(2)-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90(rsk) mutant named D2 constitutively interacts with p42(mpk1). In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42(mpk1) docking site. D2 expression uncouples the activation of p42(mpk1) and p34(cdc2)/cyclin B in response to progesterone but does not prevent signaling through p90(rsk). Instead, D2 interferes with a p42(mpk1)-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42(mpk1) and Plx1 during oocyte maturation is independent of p34(cdc2)/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42(mpk1) can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.

Induction of germinal vesicle breakdown in a cell-free preparation from starfish oocytes.

Incubation of isolated germinal vesicles in the homogenate from maturing starfish oocytes resulted in synchronous germinal vesicle breakdown (GVBD), and chromosome condensation and gathering within 30 min. GVBD in this cell-free system required aerobic conditions. The endogenous ATP-generation system was preserved in the homogenate and effective under aerobic conditions, and thus exogenous ATP was not added to the homogenate. Injection of the homogenate into immature starfish oocytes induced meiotic maturation without 1-methyladenine, indicating high activity of maturation-promoting factor (MPF) in the homogenate. MPF activity in the homogenate was stable for 2 h at room temperature, while it disappeared within 1 h in the supernatant prepared by centrifugation of the homogenate. This disappearance of MPF activity is regulated by cyclin B destruction, similar to that seen in vivo.

Translational recruitment of Xenopus maternal mRNAs in response to poly(A) elongation requires initiation factor eIF4G-1.

Xenopus oocytes accumulate maternal mRNAs which are then recruited to ribosomes during meiotic cell cycle progression in response to progesterone and coincident with poly(A) elongation. Prior to stimulation, most protein synthesis ( approximately 70%) does not require intact translation factor eIF4G (B. D. Keiper and R. E. Rhoads, 1997, Nucleic Acids Res. 25, 395-402). In the present study we have addressed the requirement of eIF4G in the recruitment of mRNAs during meiosis. Cleavage of eIF4G by coxsackievirus protease 2A inhibited progesterone-induced meiotic progression in 88% of the oocytes; prevented the recruitment of maternal mRNAs encoding cyclin B1, c-Mos, D7, and B9; and disrupted the association of eIF4G with poly(A)-binding protein. Poly(A) elongation, however, was not inhibited by eIF4G cleavage. Injection of MPF restored meiotic cell cycle progression to >60% of the oocytes but not the recruitment of cyclin B1 or B9 mRNA. Previously recruited maternal mRNAs were removed from polyribosomes following subsequent cleavage of eIF4G, indicating that eIF4G is required both to recruit and also to maintain maternal mRNAs on polyribosomes. The expression of a cleavage-resistant variant of human eIF4G-1 (G486E) significantly restored the ability to synthesize c-Mos in response to progesterone and to translate exogenous beta-globin mRNA, indicating that the inhibition by protease 2A is due to cleavage of eIF4G alone. These results indicate that intact eIF4G is required for the poly(A)-dependent recruitment of several maternal mRNAs (cyclin B1, c-Mos, D7, and B9) during meiotic cell cycle progression but not for the synthesis of most proteins.

Effect of phosphate on the second cleavage division of the rat embryo.

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.

Micro-injection of recombinant lysyl oxidase blocks oncogenic p21-Ha-Ras and progesterone effects on Xenopus laevis oocyte maturation.

Previous evidence suggested an anti-oncogenic role for lysyl oxidase, mainly in ras-transformed cells. Here we prove that recombinant lysyl oxidase is actually able to antagonize p21-Ha-Ras-induced Xenopus laevis oocyte maturation. Lysyl oxidase was also effective on progesterone-dependent maturation, indicating a block lying downstream of Ras. Maturation induced by activated 'maturation promoting factor', normally triggered by progesterone, was also inhibited by lysyl oxidase. Finally, lysyl oxidase did not abolish p42Erk2 phosphorylation upon maturation triggering, suggesting a block downstream of Erk2. Further investigation showed that lysyl oxidase action depends on protein synthesis and is therefore probably mediated by a newly synthesized protein.

Nuclear transplantation in mammals: remodelling of transplanted nuclei under the influence of maturation promoting factor.

Whilst the role of Maturation or M-phase Promoting Factor (MPF) as a universal M-phase regulator is well documented, much less attention has been paid to its role in nuclear transplantation experiments and especially to its influence upon remodelling of transplanted nuclei. There is currently wide acceptance that successful nuclear transplantation using differentiated nuclei is possible only in a cytoplasmic environment that is capable of inducing rapid nuclear de-differentiation to a pronuclear-like form. In this review our purpose is firstly, to outline the conditions under which such remodelling can be induced, and secondly, to extend the debate to include a consideration of whether complete nuclear remodelling is an absolute necessity for clonal development.

Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia.

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant of Chlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells of Tradescantia virginiana in which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.

MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts.

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.