APCcdh1 activity in mouse oocytes prevents entry into the first meiotic division

Fully grown mammalian oocytes maintain a prophase I germinal-vesicle stage arrest in the ovary for extended periods before a luteinizing hormone surge induces entry into the first meiotic division. Cdh1 is an activator of the anaphase-promoting complex (APC) and APCcdh1 is normally restricted to late M to early G1 phases of the cell cycle. Here, we find that APCcdh1 is active in mouse oocytes and is necessary to maintain prophase arrest.     

Analysis of cytoplasmic factors in developmental cleavage of mouse embryo

One-cell embryos from certain mouse strains were found incapable of developing beyond the 2-cell stage in vitro (2-cell block), but a microinjection of EDTA effectively overcame this block. When 2-cell arrested embryos were fused with embryos that had developed to the late 2-cell stage in vivo, the fusants developed beyond the 2-cell stage. Microinjection of cytoplasm of in vivo 2-cell embryos into 1-cell embryos also obviated the 2-cell block. Analyses of 35S-labeled embryos by 2-dimensional polyacrylamide gel electrophoresis indicated changes in synthetic protein patterns possibly related to this block.     

Maternal transmission ratio distortion at the mouse Om locus results from meiotic drive at the second meiotic division

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 x DDK) F(1) females and C57BL/6 males. Although significant lethality occurs in this backcross ( approximately 50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.     

Auxin-dependent control of cytoskeleton and cell shape regulates division orientation in the Arabidopsis embryo

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The involvement of Fyn kinase in resumption of the first meiotic division in mouse oocytes

The process of resumption of the first meiotic division (RMI) in mammalian oocytes includes germinal vesicle breakdown (GVBD), spindle formation during first metaphase (MI), segregation of homologous chromosomes, extrusion of the first polar body (PBI) and an arrest at metaphase of the second meiotic division (MII). Previous studies suggest a role for Fyn, a non-receptor Src family tyrosine kinase, in the exit from MII arrest. In the current study we characterized the involvement of Fyn in RMI. Western blot analysis demonstrated a significant, proteasome independent, degradation of Fyn during GVBD. Immunostaining of fixed oocytes and confocal imaging of live oocytes microinjected with Fyn complementary RNA (cRNA) demonstrated Fyn localization to the oocyte cortex and to the spindle poles. Fyn was recruited during telophase to the cortical area surrounding the midzone of the spindle and was then translocated to the contractile ring during extrusion of PBI. GVBD, exit from MI and PBI extrusion were inhibited in oocytes exposed to the chemical inhibitor SU6656 or microinjected with dominant negative Fyn cRNA. None of the microinjected oocytes showed misaligned or lagging chromosomes during chromosomes segregation and the spindle migration and anchoring were not affected. However, the extruded PBI was of large size. Altogether, a role for Fyn in regulating several key pathways during the first meiotic division in mammalian oocytes is suggested, particularly at the GV and metaphase checkpoints and in signaling the ingression of the cleavage furrow.     

Second meiotic division and polar body formation in mouse eggs fertilized in vitro

The second meiotic division and polar body formation in mouse eggs fertilized in vitro were observed by phase-contrast and polarizing microscopy, and recorded by time-lapse cinematography. Eggs were collected from oviducts of mice that had been superovulated by injections of PMS and HCG. Some eggs, inseminated with spermatozoa that had been collected from caudae epididymides of mature male mice and cultured for two to three hours before insemination, were observed continuously on a glass slide under a phase microscope. Other eggs were inseminated in Petri dishes in a 5% CO₂ incubator and examined every 20 minutes for 180 minutes. Compatible results in both sets of eggs showed that formation of the second polar body began 25–40 minutes after fusion of spermatozoon with the vitellus; it was completed 40–60 minutes later; anaphase II lasted approximately five minutes before the appearance of the furrow abstricting the second polar body. It is suggested that the furrowing associated with second polar body formation is guided by the same kind of forces that divide a cell mitotically.     

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster

An electron microscopic investigation of kinetochore structure during the first meiotic division in male Drosophila melanogaster is presented. The data suggest that the structure that is responsible for initial microtubule attachment and chromosome orientation is a single, bilaminar hemisphere on each half-bivalent. Following the initial attachment this structure undergoes morphogenesis to a double-disc structure that reflects the underlying duality of sister chromatids in the half-bivalent. Thus these data support Darlington's idea that sister chromatids disjoin to the same spindle pole because they share a single kinetochore. Additionally, these data suggest that the meiotic mutations ord and mei-S332 sometimes cause premature “doubling” of the kinetochore region though, as discussed, possibly for a trivial reason.     

Mitotic spindles and cleavage planes are oriented randomly in the two-cell mouse embryo

Most experimental embryological studies performed on the early mouse embryo have led to the conclusion that there are no mosaically distributed developmental determinants in the zygote and early embryo (for example see [1-6]). It has been suggested recently that "the cleavage pattern of the early mouse embryo is not random and that the three-dimensional body plan is pre-patterned in the egg" (in [7] for review see [8-10]). Two major spatial cues influencing the pattern of cleavage divisions have been proposed: the site of the second meiotic division [11, 12] and the sperm entry point [13-14], although the latter is controversial [15-17]. An implication of this hypothesis is that the orientations of the first few cleavage divisions are stereotyped. Such a define cleavage pattern, leading to the segregation of developmental determinants, is observed in many species [18]. Recently, it was shown that the first cleavage plane is not predetermined but defined by the topology of the two apposing pronuclei [19]. Because the position of the female pronucleus is dependent upon the site of polar body extrusion and the position of the male pronuclei is dependent upon the sperm entry point [19-20], this observation leaves open the possibility that the sperm may provide some kind of directionality [7]. But, even if asymmetries were set up only after fertilization, a stereotyped cleavage pattern should take place during the following cleavage divisions. Thus, we studied the cleavage pattern of two-cell embryos by videomicroscopy to distinguish between the two hypotheses. After the mitotic spindle formed, its orientation did not change until cleavage. During late metaphase and anaphase, the spindle poles appear to be anchored to the cortex through astral microtubules and PARD6a. Only at the time of cleavage, during late anaphase, do the forming daughter cells change their relative positions. These studies show that cleavage planes are oriented randomly in two-cell embryos. This argues against a prepatterning of the mouse embryo before compaction.     

First cleavage of the mouse embryo responds to change in egg shape at fertilization

Although mouse development is regulative, the cleavage pattern of the embryo is not random. The first cleavage tends to relate to the site of the previous meiosis. Sperm entry might provide a second cue, but evidence for and against this is indirect and has been debated. To resolve whether sperm entry position relates to the first cleavage, we have followed development from fertilization by time-lapse imaging. This directly showed cytokinesis passes close to the site of the previous meiosis and to both the sperm entry site and trajectory of the male pronucleus in a significant majority of eggs. We detected asymmetric distribution of Par6 protein in relation to the site of meiosis, but not sperm entry. Unexpectedly, we found the egg becomes flattened upon fertilization in an actin-mediated process. The sperm entry position tends to lie at one end of the short axis along which cleavage will pass. When we manipulated eggs to change their shape, this repositioned the cleavage plane such that eggs divided along their experimentally imposed short axis. Such manipulated eggs were able to develop to term, emphasizing the regulative nature of their development.     

Effects of nitrous oxide on preimplantation mouse embryo cleavage and development

Preimplantation mouse embryos were exposed to nitrous oxide for 30 min to determine its effects on subsequent development after short durations of exposure. Two-cell mouse embryos were exposed to 60% nitrous oxide/40% oxygen at 6-7 h, 3-4 h, or 0-1 h prior to the expected onset of their first cleavage in vitro, or at the 4-cell or morula stages. Effects of nitrous oxide were not observed except in 2-cell embryos treated within 4 h of the expected in vitro cleavage. At 3-4 h and 0-1 h prior to the onset of cleavage, exposure to 60% nitrous oxide/40% oxygen resulted in blastocyst development rates of 27.7% and 4.7%, respectively, while control rates ranged from 75% to 77%. The majority of affected embryos were halted at the 2-cell stage before completing cell division. Similar effects were obtained with 80% nitrous oxide/20% oxygen. Thus, we conclude that brief exposure of mouse preimplantation embryos to nitrous oxide may be deleterious to subsequent embryo cleavage, but this effect is highly dependent on the developmental stage at which exposure occurs.     

Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development

Two independent studies have recently suggested similar models in which the embryonic and abembryonic parts of the mouse blastocyst become separated already by the first cleavage division. However, no lineage tracing studies carried out so far on early embryos provide the support for such a hypothesis. Thus, to re-examine the fate of blastomeres of the two-cell mouse embryo, we have undertaken lineage tracing studies using a non-perturbing method. We show that two-cell stage blastomeres have a strong tendency to develop into cells that comprise either the embryonic or the abembryonic parts of the blastocyst. Moreover, the two-cell stage blastomere that is first to divide will preferentially contribute its progeny to the embryonic part. Nevertheless, we find that the blastocyst embryonic-abembryonic axis is not perfectly orthogonal to the first cleavage plane, but often shows some angular displacement from it. Consequently, there is a boundary zone adjacent to the interior margin of the blastocoel that is populated by cells derived from both earlier and later dividing blastomeres. The majority of cells that inhabit this boundary region are, however, derived from the later dividing two-cell stage blastomere that contributes predominantly to the abembryonic part of the blastocyst. Thus, at the two-cell stage it is already possible to predict which cell will contribute a greater proportion of its progeny to the abembryonic part of the blastocyst (region including the blastocyst cavity) and which to the embryonic part (region containing the inner cell mass) that will give rise to the embryo proper.     

Organisation and assembly of the surface membrane during early cleavage of the mouse embryo

Summary An attempt was made to understand the ways in which ‘newly inserted’ membrane was organised in relation to existing membrane during early cleavage of the mouse embryo by (i) monitoring the redistribution of a variety of surface-binding ligands (applied to the embryo during the previous cell cycle) and (ii) analysing the localisation of newly synthesised lipid at defined stages during the second cell cycle. The membrane dynamics of the embryo appear similar to those of somatic cells during cytokinesis and/or motility, and are consistent with previous suggestions (Pratt 1985) that the main cytocortical domains of the polarised 8-cell blastomere may start to diverge during early cleavage as a result of localised assembly and reorganisation of the embryo cytocortex.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

Control of the meiotic cell division program in plants

While the question of why organisms reproduce sexually is still a matter of controversy, it is clear that the foundation of sexual reproduction is the formation of gametes with half the genomic DNA content of a somatic cell. This reduction in genomic content is accomplished through meiosis that, in contrast to mitosis, comprises two subsequent chromosome segregation steps without an intervening S phase. In addition, meiosis generates new allele combinations through the compilation of new sets of homologous chromosomes and the reciprocal exchange of chromatid segments between homologues. Progression through meiosis relies on many of the same, or at least homologous, cell cycle regulators that act in mitosis, e.g., cyclin-dependent kinases and the anaphase-promoting complex/cyclosome. However, these mitotic control factors are often differentially regulated in meiosis. In addition, several meiosis-specific cell cycle genes have been identified. We here review the increasing knowledge on meiotic cell cycle control in plants. Interestingly, plants appear to have relaxed cell cycle checkpoints in meiosis in comparison with animals and yeast and many cell cycle mutants are viable. This makes plants powerful models to study meiotic progression and allows unique modifications to their meiotic program to develop new plant-breeding strategies.     

Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.     

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.