Meiotic maternal chromosomes introduced to the late mouse zygote are recruited to later embryonic divisions

The aim of this study was to investigate the fate of an additional female genome introduced to a dividing zygote. Maternal chromatin in the form of karyoplasts containing a metaphase II spindle were fused to zygotes blocked in anaphase or telophase of the first cleavage. Permanent preparations made 20-40 min after fusion at anaphase revealed that the donor maternal chromosomes had entered anaphase or telophase in 16 out of 18 cases. A further two groups of embryos that were fused at either anaphase or anaphase/telophase were cultured to the first division. Division occurred 50 min after fusion in both groups of embryos (86 and 85.1%, respectively), of which most divided to two cells (80 and 71.6% of total) and the remainder divided to three cells. About two thirds of two-cell embryos contained an extra nucleus in one blastomere. Nuclei containing donor maternal chromosomes reached a similar size to recipient nuclei in 68% of embryos derived from anaphase-blocked zygotes, in contrast to 31.1% of embryos derived from anaphase/telophase-blocked embryos. Replication of DNA in donor nuclei closely followed the timing and intensity of that in control embryos. When fixed 24 hr after fusion, one third of embryos were still at the two-cell stage, with one or both blastomeres showing a single metaphase plate of the second cleavage. In the remaining embryos, three or four cells were present, some containing two nuclei. Blastocysts developed in 50% of fused embryos and three young were born after transfer of cleaving hybrid embryos to recipients. Chromosome preparations from bone marrow of the young contained 3-4 tetraploid metaphase plates per several hundred plates counted compared with none in control embryos. In conclusion, additional maternal chromosomes can be introduced at the late-dividing zygote and join the embryonic cell cycles during subsequent divisions. This method may provide a useful approach for studying changes specific to the maternal genome during early cell cycles of the mammalian embryo.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

First cell fate decisions and spatial patterning in the early mouse embryo

A growing body of evidence indicates that although the early mouse embryo retains flexibility in responding to perturbations, its patterning is initiated at the earliest developmental stages. There are a few spatial cues that are able to influence the pattern of cleavage divisions: one of these lies in the vicinity of the previous meiotic division, the second is associated with the sperm entry and, related to this, the third is the cell shape. Furthermore, the first cleavage separates the zygote into two cells that tend to follow distinguishable fates: one contributes mainly to the embryonic part of the blastocyst, and the other to the abembryonic. The cumulative effect of the early asymmetries generated through cleavage might lead to asymmetric interactions between the first lineages of cells. This could influence development of patterning after implantation. These early polarity cues serve to bias patterning and not as definitive determinants.     

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.     

Unique Pattern of ORC2 and MCM7 Localization During DNA Replication Licensing in the Mouse Zygote

In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.     

Radiosensitivity variation during the cell cycle in pronuclear mouse embryos in vitro

Abstract. The radiosensitivity of pronuclear mouse (B6D2 F1 x ICR) embryos has been measured in vitro as a function of time during the cell cycle. This was done by measuring the dose of X-rays (LD50) required to prevent development of 50% of the pronuclear embryos to the blastocyst stage in 5 days of culture. The LD50 was found to vary from 1 to 2 Gy during the period from G1 to the first cleavage. The cell cycle in the pronuclear embryo was analysed by [³H]thymidine autoradiography. Compared with earlier studies on two-cell mouse embryo radiosensitivity, the pronuclear embryos appear to be more sensitive to radiation than the two-cell embryos. If, however, one considers the radiation sensitivity on a blastomere basis, the pronuclear embryos are not different in their radiation sensitivity from the two-cell embryos. Thus, during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo.     

Kinetochore appearance during meiosis,fertilization and mitosis in mouse oocytes and zygotes

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pronuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.     

Wnt-dependent spindle polarization in the early C. elegans embryo

Correct orientation of the mitotic spindle is crucial for the proper segregation of localized determinants and the correct spatial organization of cells in early embryos. The cues dividing cells use to orient their mitotic spindles are currently the subject of intensive investigation in a number of model systems. One of the cues that cells use during spindle orientation is provided by components of the Wnt signaling pathway. Because of its stereotypical cleavage divisions, the availability of Wnt pathway mutants and the ability to perform RNAi, and because cell-cell interactions can be studied in vitro, the C. elegans embryo continues to be a useful system for identifying specific cell-cell interactions in which Wnt-dependent signals polarize the mitotic spindle. This review discusses the evidence for involvement of Wnt signaling during spindle orientation in several contexts in the early C. elegans embryo, a process that involves upstream Wnt effectors but does not involve downstream nuclear effectors of Wnt signaling, and places this Wnt spindle orientation pathway in the larger context of other known modulators of spindle orientation in animal embryos.     

Seasonal variation in cell cycle during early development of the mouse embryo.

Studies of the cell cycle of mouse embryos before implantation were conducted using Giemsa and DAPI stains. The time of embryo recovery did not affect the success rate of cultures during the winter, but embryos cultured during the summer showed the 'two-cell block' phenomenon at the early two-cell stage, 30-37 h after the injection of human chorionic gonadotrophin. There was no significant difference in the number of embryos collected per mouse between summer and winter, but cleavage from the two-cell to the four-cell stage occurred later in the summer than in the winter. Cell cycle of mouse embryos may therefore show seasonal variation.     

Full-term development of mouse eggs transplanted with male pronuclei derived from round spermatids: the effect of synchronization between male and female pronucleus on embryonic development

Pronucleus transplanted mice have been produced, but their donor male pronuclei were derived from mature sperm and were completely synchronous with female pronuclei because both male and female pronuclei came from the same fertilized oocyte. The present study firstly produced male pronuclei by introducing round spermatids into enucleated mouse oocytes, then transferred the male pronuclei into mouse oocytes at three activation stages and finally compared the effect of three kinds of oocytes on the development of reconstructed embryos. Our results indicate that, in enucleated oocytes, mouse round spermatid nuclei can transform to male pronuclei in a higher proportion, and the synchronization between male and female pronucleus does not significantly influence the early cleavage but the later and full-term development of reconstructed embryos.     

Orientation of Mitotic Spindles during the 8- to 16-Cell Stage Transition in Mouse Embryos

Background

Asymmetric cell divisions are involved in the divergence of the first two lineages of the pre-implantation mouse embryo. They first take place after cell polarization (during compaction) at the 8-cell stage. It is thought that, in contrast to many species, spindle orientation is random, although there is no direct evidence for this.

Methodology/Principal Findings

Tubulin-GFP and live imaging with a spinning disk confocal microscope were used to directly study spindle orientation in whole embryos undergoing the 8- to 16-cell stage transition. This approach allowed us to determine that there is no predetermined cleavage pattern in 8-cell compacted mouse embryos and that mitotic spindle orientation in live embryo is only modulated by the extent of cell rounding up during mitosis.

Conclusions

These results clearly demonstrate that spindle orientation is not controlled at the 8- to 16-cell transition, but influenced by cell bulging during mitosis, thus reinforcing the idea that pre-implantation development is highly regulative and not pre-patterned.     

Early developmental processes in the fertilised honeybee (Apis mellifera) oocyte

The behaviour of sperm from egg penetration until creation of the zygote, the development of the maternal pronucleus, and the two first cleavage divisions were studied by use of fluorescence microscopy. It was found that 4-12 sperm penetrate the egg membranes prior to oviposition. Contrary to previous reports, we found that only 1-7 sperm move from their initial location just beneath the vitelline membrane and into the cytoplasm, where they develop into paternal pronuclei. At the time of oviposition, the oocyte nucleus was usually at the stage of metaphase I, rather than anaphase I as previously reported. At 26+/-2.5 minutes the meiotic process had entered the stage of metaphase II. The paternal and maternal pronuclei formed at 55+/-2.6 minutes, and they fused at 93+/-7.3 minutes. The mitotic division of the zygote was completed at 119+/-6.5 minutes.     

Mutations in ooc-5 and ooc-3 disrupt oocyte formation and the reestablishment of asymmetric PAR protein localization in two-cell Caenorhabditis elegans embryos.

The early development of Caenorhabditis elegans embryos is characterized by a series of asymmetric divisions in which the mitotic spindle is repeatedly oriented on the same axis due to a rotation of the nuclear-centrosome complex. To identify genes involved in the control of spindle orientation, we have screened maternal-effect lethal mutants for alterations in cleavage pattern. Here we describe mutations in ooc-5 and ooc-3, which were isolated on the basis of a nuclear rotation defect in the P(1) cell of two-cell embryos. These mutations are novel in that they affect the asymmetric localization of PAR proteins at the two-cell stage, but not at the one-cell stage. In wild-type two-cell embryos, PAR-3 protein is present around the entire periphery of the AB cell and prevents nuclear rotation in this cell. In contrast, PAR-2 functions to allow nuclear rotation in the P(1) cell by restricting PAR-3 localization to the anterior periphery of P(1). In ooc-5 and ooc-3 mutant embryos, PAR-3 was mislocalized around the periphery of P(1), while PAR-2 was reduced or absent. The germ-line-specific P granules were also mislocalized at the two-cell stage. Mutations in ooc-5 and ooc-3 also result in reduced-size oocytes and embryos. However, par-3 ooc double-mutant embryos can exhibit nuclear rotation, indicating that small size per se does not prevent rotation and that PAR-3 mislocalization contributes to the failure of rotation in ooc mutants. We therefore postulate that wild-type ooc-5 and ooc-3 function in oogenesis and in the reestablishment of asymmetric domains of PAR proteins at the two-cell stage.     

Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments

Female meiosis in higher organisms consists of highly asymmetric divisions, which retain most maternal stores in the oocyte for embryo development. Asymmetric partitioning of the cytoplasm results from the spindle's "off-center" positioning, which, in mouse oocytes, depends mainly on actin filaments [1, 2]. This is a unique situation compared to most systems, in which spindle positioning requires interactions between astral microtubules and cortical actin filaments [3]. Formin 2, a straight-actin-filament nucleator, is required for the first meiotic spindle migration to the cortex and cytokinesis in mouse oocytes [4, 5]. Although the requirement for actin filaments in the control of spindle positioning is well established in this model, no one has been able to detect them in the cytoplasm [6]. Through the expression of an F-actin-specific probe and live confocal microscopy, we show the presence of a cytoplasmic actin meshwork, organized by Formin 2, that controls spindle migration. In late meiosis I, these filaments organize into a spindle-like F-actin structure, which is connected to the cortex. At anaphase, global reorganization of this meshwork allows polar-body extrusion. In addition, using actin-YFP, our FRAP analysis confirms the presence of a highly dynamic cytoplasmic actin meshwork that is tightly regulated in time and space.     

Asymmetric division in mouse oocytes: with or without Mos

In both vertebrates and invertebrates, meiotic divisions in oocytes are typically asymmetric, resulting in the formation of a large oocyte and small polar bodies. The size difference between the daughter cells is usually a consequence of asymmetric positioning of the spindle before cytokinesis. Spindle movements are often related to interactions between the cell cortex and the spindle asters [1,2]. The spindles of mammalian oocytes are, however, typically devoid of astral microtubules, which normally connect the spindle to the cortex, suggesting that another mechanism is responsible for the unequal divisions in these oocytes. We observed the formation of the first polar body in wild-type oocytes and oocytes derived from c-Mos knockout mice [3]. In wild-type oocytes, the meiotic spindle formed in the centre of the cell and migrated to the cortex just before polar-body extrusion. The spindle did not elongate during anaphase. In mos-/- oocytes, the spindle formed centrally but did not migrate, although an asymmetric division still took place. In these oocytes, the spindle elongated during anaphase and the pole closest to the cortex moved while the other remained in place. Thus, a compensation mechanism exists in mouse oocytes and formation of the first polar body can be achieved in two ways: either after migration of the spindle to the cortex in wild-type oocytes, or after elongation, without migration, of the first meiotic spindle in mos-/- oocytes.     

Appearance and heterochromatin localization of HP1α in early mouse embryos depends on cytoplasmic clock and H3S10 phosphorylation

Pericentric constitutive heterochromatin surrounds centromeric regions and is important for centromere function and chromatid cohesion. HP1 (heterochromatin protein 1), a homolog of yeast Swi6, has been shown to be indispensible for proper heterochromatin structure and function. In mammalian somatic cells, two HP1 isoforms, HP1α and HP1β, are constitutively present in pericentric heterochromatin until late G₂, when they dissociate from heterochromatin. Subsequently, they re-associate with heterochromatin at late anaphase. In one-cell mouse embryos, pericentric heterochromatin has a unique configuration and features. It does not form heterochromatin clusters observed in somatic cells and known as chromocenters. Instead, in both pronuclei, it surrounds nucleolar precursor bodies (NBPs), forming ring-like structures. These regions contain HP1β but lack HP1α in both pronuclei. In subsequent interphases, HP1β is constitutively found in heterochromatin until the blastocyst stage. It is not known when HP1α appears and what is its function in early mouse embryos. Here, we show that HP1α appears for the first time at late S phase of two-cell stage, at the time when pericentric heterochromatin is replicated. Its appearance is regulated at the level of translation. In two-cell embryos, the amount of HP1α that can bind to these regions is regulated by phosphorylation of serine 10 of histone H3 (H3S10Ph). Elimination of HP1α by siRNA interfered with centromere relocation from heterochromatin surrounding NPBs to pro-chromocenters at the two-cell stage but did not affect preimplantation develoment to the blastocyst stage.