Cytoplasmic control of nuclear maturation in mouse oocytes

Oocytes with germinal vesicles were cut into anucleate and nucleate fragments. At the time of germinal vesicle breakdown (GVBD) in nucleate fragments (after 2–3 h of culture) sister anucleate fragments were fused with the help of inactivated Sendai virus with interphase blastomeres from 2-cell embryos. The hybrid cells were examined after h and 20 h. The anucleate fragments induced chromosome condensation in the nuclei of interphase blastomeres immediately after fusion. On this basis it may be concluded that GVBD and nuclear maturation in mouse oocytes is induced by a cytoplasmic factor which is produced or unmasked independently of the nucleus.     

Control of microtubule nucleating activity in the cytoplasm of maturing mouse oocytes.

Taxol, a drug which promotes microtubule assembly, was used to assess the microtubule nucleating activity of pericentriolar material (PCM) in mouse oocytes prevented from undergoing germinal vesicle breakdown (GVBD), compared with oocytes allowed to proceed normally through GVBD and also in nucleate and anucleate oocyte fragments. Both immunofluorescence staining and ultrastructural analysis reveal that taxol induces aster formation in the cortex of oocytes undergoing GVBD, while formation of a continuous sheet of microtubule bundles parallel to the membrane is induced in metabolically GV-arrested oocytes. Since taxol also induces the formation of asters in anucleate as well as in nucleate oocyte fragments, provided they are not treated with activators of protein kinases A or C, it is concluded that microtubule nucleating activity is related to the acquisition of Maturation Promoting Factor (MPF) and does not require mixing between the nucleoplasm and cytoplasm.     

Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes

We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.     

Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusions

Mouse and porcine fully grown oocytes at metaphase I(MI) were fused to one or more fully grown oocytes of the same species that contained an intact germinal vesicle (GV). In fused cells containing one GV, premature chromosome condensation (PCC) was observed. In fused cells containing more than one GV, germinal vesicle breakdown (GVBD) and PCC were delayed. Fusion of an MI fully grown oocyte with a growing oocyte resulted in rapid PCC, whereas, fusion of an MI fully grown oocyte with more than one growing oocyte resulted in neither PCC nor GVBD. Moreover, MI chromosomes formed a clump of chromatin. Results of these experiments suggest that the delay in GVBD in fusions of MI oocytes with multiple GV-intact oocytes was due to dilution of maturation promoting factor (MPF) by the cytoplasm of the GV-intact oocytes and that the cytoplasm of growing oocytes can inhibit MPF present in MI oocytes.     

Two phases of calcium requirement during starfish meiotic maturation

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.     

小鼠卵母细胞体外成熟不同阶段对钙的依赖性

To determine the role of calcium and calmodulin in mouse oocyte maturation, we examined the distribution of intracellular calcium during mouse oocyte maturation by using Mira Cal Imaging System. The calcium was present homogeneously in oocytes with intact germinal vesicle (GV) and accumulated around the nuclear region after GV breakdown(GVBD). The high level of calcium disappeared 6 hours later after GVBD. In the presence of 50 mumol/L BAPTA/AM, we failed to observe this phenomena. All eggs treated with 20 mumol/L W7, an antagonist of calmodulin, 50 mumol/L BAPTA/AM, a calcium chelator, could not develop to metaphase II (MII), although GVBD was not affected. We also detected the activity of a cytoplasmic maturation-promoting factor (MPF). W7 and BAPTA/AM had no effects on the rise of MPF activity in the course of maturation. We suggest that compartment distribution of calcium around nuclear region plays an important role in mouse oocyte maturation.     

The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes.

Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase-telophase transition, or (3) at metaphase II were fused to germinal vesicle-staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase I oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re-enters metaphase.     

The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis

Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.     

Control of chromosome behavior in amphibian oocytes. I. The activity of maturing oocytes inducing chromosome condensation in transplanted brain nuclei

The capability of oocyte cytoplasm to induce chromosome condensation was studied by transplantation of isolated brain nuclei into Rana pipiens oocytes induced to undergo maturation in vitro by progesterone treatment. It was found that the chromosome condensation activity (CCA) first appeared in the cytoplasm of maturing oocytes shortly after germinal vesicle breakdown (GVBD), persisted in fully mature oocytes, but rapidly disappeared when the oocytes were artificially activated. A comparison of the time course of the oocyte chromosome condensation cycle and of brain chromosome condensation in maturing and activated oocytes revealed a close temporal correlation between the two, suggesting that both are under the control of the same cytoplasmic factor(s). Oocytes enucleated before GVBD always failed to develop CCA. The CCA could be restored in enucleated oocytes by injecting nucleoplasm obtained from oocytes that had not yet undergone GVBD although this same nucleoplasm was incapable of producing CCA when mixed with the cytoplasm of oocytes that had not reached the stage of GVBD. It was therefore suggested that the CCA had a dual origin involving both cytoplasmic maturation and GV materials.     

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.     

Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis

We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material.     

Fer tyrosine kinase is required for germinal vesicle breakdown and meiosis-I in mouse oocytes

The control of microtubule and actin-mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Our previous studies demonstrated a role for FYN tyrosine kinase in chromosome and spindle organization and in cortical polarity of the mature mammalian oocyte. In addition to Fyn, mammalian oocytes express the protein tyrosine kinase Fer at high levels relative to other tissues. The objective of the present study was to determine the function of this kinase in the oocyte. Feline encephalitis virus (FES)-related kinase (FER) protein was uniformly distributed in the ooplasm of small oocytes, but became concentrated in the germinal vesicle (GV) during oocyte growth. After germinal vesicle breakdown (GVBD), FER associated with the metaphase-I (MI) and metaphase-II (MII) spindles. Suppression of Fer expression by siRNA knockdown in GV stage oocytes did not prevent activation of cyclin dependent kinase 1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to GVBD or during MI. The resultant phenotype displayed condensed chromosomes trapped in the GV, or condensed chromosomes poorly arranged in a metaphase plate but with an underdeveloped spindle microtubule structure or chromosomes compacted into a tight sphere. The results demonstrate that FER kinase plays a critical role in oocyte meiotic spindle microtubule dynamics and may have an additional function in GVBD.     

Nuclear maturation in pig and rabbit oocytes after interspecific fusion

Porcine ovarian oocytes were fused with either homologous (porcine) or heterologous (rabbit) oocytes, both at different stages of maturation. The maturation-promoting factor (MPF) present in maturing porcine oocytes or ovulated rabbit oocytes induced rapid chromosome condensation of the oocytes with intact germinal vesicles (GVs). In the case of activation of ovulated rabbit oocyte, germinal vesicle breakdown (GVBD) of porcine oocytes was incomplete or did not occur. In the giant cells consisting of two immature porcine oocytes, meiotic maturation proceeded in the same manner as in unfused oocytes. However, in cells derived from fusion of immature porcine and rabbit oocytes, two metaphase groups of chromosomes were observed 6 h after fusion. It may be concluded that GVBD is governed after fusion by the cytoplasm originating from the oocytes of more advanced stages of maturation or from those which mature faster.     

Chk2 Regulates Cell Cycle Progression during Mouse Oocyte Maturation and Early Embryo Development

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein γ-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo 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.     

Requirements of Src family kinase during meiotic maturation in mouse oocyte

The Src family kinase (SFK) is important in normal cell cycle control. However, its role in meiotic maturation in mammalian has not been examined. We used confocal microscope immunofluorescence to examine the in vitro dynamics of the subcellular distribution of SFK during the mouse oocyte meiotic maturation and further evaluated the functions of SFK via biochemical analysis using a specific SFK pharmacological inhibitor, PP(2). Our results showed that nonphospho-SFK was absent in oocyte upon its release from follicle. Nonphospho-SFK appeared in cytoplasm 0.5 hr after the release of oocyte and translocated to germinal vesicle (GV) before germinal vesicle breakdown (GVBD). After GVBD, nonphospho-SFK colocated with condensed chromosomes. In occyte at metaphase I (MI) and telophase I, nonphospho-SFK accumulated in the cortex and the cleavage furrow respectively besides its existence in cytoplasm in both stages. In oocyte at metaphase II (MII), nonphospho-SFK concentrated at the aligned chromosomes. In contrast, phospho-SFK was absent in oocyte until 1 hr after its release from the follicle. Phospho-SFK accumulated in the GV, the cortex, and cytoplasm immediately prior to GVBD. After GVBD, phospho-SFK evenly distributed in oocyte. In oocyte at MII, phospho-SFK localized throughout the cytoplasm and under the egg member. When the SFK activity was inhibited, the oocyte failed to initiate GVBD, could not go into MII, and could not extrude the first polar body. Our results demonstrated that SFK is required for meiotic maturation in mouse oocyte.     

Role of Greatwall kinase in release of mouse oocytes from diplotene arrest

In eukaryotes, mitosis entry is induced by activation of maturation‐promoting factor (MPF), which is regulated by a network of kinases and phosphatases. It has been suggested that Greatwall (GWL) kinase was crucial for the M‐phase entry and could maintain cyclin B–Cdc2 activity through regulation of protein phosphatase 2A (PP2A), a counteracting phosphatase of MPF. Here, the role of GWL was assessed during release of mouse oocytes from prophase I arrest. GWL was crucial for meiotic maturation in mouse oocytes. As a positive regulator for meiosis resumption, GWL was continually expressed in germinal vesicle (GV) and MII stage oocytes and two‐cell stage embryos. Additionally, GWL localized to the nucleus and dispersed into cytoplasm during GV breakdown (GVBD). Furthermore, downregulation of GWL or overexpression of catalytically‐inactive GWL inhibited partial meiotic maturation. This prophase I arrest induced by GWL depletion could be rescued by the PP2A inhibition. However, both GWL‐depleted and rescued oocytes had severe spindle defects that hardly reached MII. In contrast, oocytes overexpressing wild‐type GWL resumed meiosis and progressed to MII stage. Thus, our data demonstrate that GWL acts in a pathway with PP2A which is essential for prophase I exit and metaphase I microtubule assembly in mouse oocytes.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.     

The role of RhoA in the germinal vesicle breakdown of mouse oocytes

We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADP-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVBD = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-l-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes.