Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam (Spisula solidissima) oocytes

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.     

Interaction of cell cycle kinases, microtubules, and chromatin in ascidian oocytes during meiosis

We used kinase assays and confocal microscopy to study the interaction of cell cycle proteins with microtubule organising centres (MTOC) and chromatin in ascidian oocytes during meiosis. The activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) appear not to be correlated in control oocytes. MPF activity peaks during metaphase I and II of the meiotic cell cycle whereas the activity of MAPK peaks at telophase I and is subsequently degraded to remain at low levels for the remainder of meiosis. The protein synthesis inhibitor emetine induces the degradation in MPF activity in unfertilized metaphase-I (M-I) oocytes, while MAPK is unaffected. Emetine does not alter the activities of these cell cycle kinases in fertilized oocytes during meiosis I but MPF activity remains low while MAPK activity is high for an elongated time period and oocytes do not complete meiosis I. Emetine induces maternal MTOC duplication in unfertilized M-I oocytes and prevents sperm aster growth in fertilized oocytes, but it does not alter the M-I meiotic apparatus in unfertilized oocytes. These experiments suggest that neither MPF alone nor emetine-sensitive proteins are responsible for M-I arrest in ascidian oocytes, MAPK may ensure this stability. In addition, we showed that the maternal MTOC is present at M-I but suppressed from duplicating in an emetine-sensitive manner.     

Modulation of cell cycle control during oocyte‐to‐embryo transitions

Ex ovo omnia—all animals come from eggs—this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII‐arrest), the first mitotic cell cycle, and early embryonic divisions.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.     

Evolution of the acquisition of fertilization competence and polyspermy blocks during meiotic maturation

In many animals, fully grown oocytes are arrested at prophase of meiosis I. Before or after ovulation/spawning, a secondary arrest occurs at metaphase of meiosis I or II (MI or II, respectively). MI arrest in the ovary is released after spawning, and is followed by fertilization, whereas MI and MII arrest after ovulation are released by fertilization. Insemination of isolated oocytes from the ovaries at an inappropriate time increases the rate of polyspermy, indicating that ovaries provide the proper environment for acquisition of the polyspermy blocks and the development of competence to be fertilized normally. Due to MI arrest in the ovaries or MI/MII arrest after ovulation/spawning, the fertilizable period can be elongated. Thus, MI and MII arrest may play a role in maintaining the cell-cycle phases to enable normal fertilization. Here, the evolution of fertilization timing is discussed.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.     

A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes

Precise coordination of meiotic progression is a critical determinant of an egg's capacity to be fertilized successfully, and zinc has emerged as a key regulatory element in this process. An early manifestation of a regulatory role for this transition metal is the significant increase in total intracellular zinc. This accumulation is essential for meiotic progression beyond telophase I and the establishment of meiotic arrest at metaphase II. The subsequent developmental event, fertilization, induces a rapid expulsion of labile zinc that is a hallmark event in meiotic resumption. In the present study, we show that the zinc fluxes work, in part, by altering the activity of the cytostatic factor (CSF), the cellular activity required for the establishment and maintenance of metaphase II arrest in the mature, unfertilized egg. We propose a model in which zinc exerts concentration-dependent regulation of meiosis through the CSF component EMI2, a zinc-binding protein. Together, the data support the conclusion that zinc itself, through its interaction with EMI2, is a central component of the CSF.     

Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes.     

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

A metaphase pause: Hormone-induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the 'acceleration' of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1–methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisaster oocytes pauses transiently at metaphase I for more than 40min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term 'metaphase pause' is proposed to define this long duration of metaphase I in starfish oocytes.     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

Effects of spermatozoa during in vitro meiosis progression in the porcine germinal vesicle oocytes

We have investigated the effect of co-culture with porcine spermatozoa on in vitro maturation of porcine germinal vesicle (GV) oocytes before fertilization. Most oocytes were arrested at the first prophase of meiosis when oocytes were cultured in TCM 199 alone, but the proportion of oocytes that reached metaphase II was significantly elevated by co-incubation with spermatozoa in vitro. The oocyte maturation effect was observed with intact and parts of spermatozoa (head and tail) collected from adult swine (regardless of source). However, gonocytes from the newborn porcine testis were not able to enhance in vitro maturation of porcine germinal vesicle oocytes. Interestingly, the oocyte maturation effect by spermatozoa was not decreased with heat treatment, but the maturation effect of oocyte treatment disappeared with exposure to detergent in sperm suspension. Porcine spermatozoa were also observed to stimulate meiosis of oocytes, which was maintained at meiotic arrest using dibutyryl cyclic AMP or forskolin. The study suggests that (i) membrane of porcine spermatozoa contains a substance(s) that can enhance in vitro maturation of oocytes prior to fertilization, (ii) the putative meiosis-enhancing substance(s) of spermatozoa from adult testes retains the oocyte maturation effect during transportation of spermatozoa through epididymis, and (iii) the putative meiosis-enhancing substance(s) is able to overcome the inhibitory effect of dibutyryl cyclic AMP or forskolin by inducing germinal vesicle breakdown of porcine cumulus-oocyte complexes maintained in meiotic arrest.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

The anaphase-promoting complex/cyclosome inhibitor Emi2 is essential for meiotic but not mitotic cell cycles

Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown. Here we show that during oocyte maturation, Emi2 appears only after metaphase I, and its level peaks at CSF arrest (metaphase II). In M phase, Emi2 undergoes a phosphorylation-dependent electrophoretic shift. Microinjection of antisense oligonucleotides against Emi2 into stage VI oocytes blocks progression through meiosis II and the establishment of CSF arrest. Recombinant Emi2 rescues CSF arrest in these oocytes and also causes CSF arrest in egg extracts and in blastomeres of two-cell embryos. Fertilization triggers rapid, complete degradation of Emi2, but it is resynthesized in the first embryonic cell cycle to reach levels 5-fold lower than during CSF arrest. However, depletion of the protein from cycling egg extracts does not prevent mitotic cell cycle progression. Thus, Emi2 plays an essential role in meiotic but not mitotic cell cycles.     

Developmental control of oocyte maturation and egg activation in metazoan models

Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.     

Developmental defects observed in hypomorphic anaphase-promoting complex mutants are linked to cell cycle abnormalities

In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis II altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects.