Oocyte Polarity Research Articles

Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster.

The nuage is an electron-dense perinuclear structure that is known to be a hallmark of animal germ-line cells. Although the conservation of the nuage throughout evolution accentuates its essentiality, its role(s) and the exact mechanism(s) by which it functions in the germ line still remain unknown. Here, we report a nuage component, Krimper (KRIMP), in Drosophila melanogaster and show that it ensures the repression of the selfish genetic elements in the female germ line. The Krimp loss-of-function allele exhibited female sterility, defects in karyosome formation and oocyte polarity, and precocious osk translation. These phenotypes are commonly observed in the other nuage component mutants, vasa (vas) and maelstrom (mael), and the RNA-silencing component mutants, spindle-E (spn-E) and aubergine (aub), suggesting a shared underlying defect that uses RNA silencing. Moreover, we demonstrated that the localization of the nuage components depends on both SPN-E and AUB and that the selfish genetic elements were derepressed to different extents in the nuage component mutants, as well as in aub and armitage (armi) mutants. In the nuage component mutants, vas, krimp, and mael, the levels of roo, I-element, and HeT-A repeat-associated small interfering RNAs were greatly reduced. Hence, our data suggest that the nuage functions as a specialized center that protects the genome in the germ-line cells via gene regulation mediated by repeat-associated small interfering RNAs.

Establishment of oocyte polarity in vertebrates

Establishment of oocyte polarity in vertebrates

The Ran GTPase Mediates Chromatin Signaling to Control Cortical Polarity during Polar Body Extrusion in Mouse Oocytes

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.

Marsupial oocytes, fertilization and embryonic development can provide useful tools to study developmental mechanisms

An analysis of several features of marsupial oocytes and developmental stages from the zygote to the neonate shows the advantages of studying these to answer questions about mammalian development in general. Marsupials make good model systems providing that species have well established breeding and husbandry protocols, a detailed time table of embryonic development associated with appropriate monitoring to collect desired stages and an induced ovulation protocol to allow collection of normal oocytes and developmental stages. One of the specific features of marsupial development that would provide further insight into problems in mammalian development is distinct oocyte and/or zygote polarity. Additionally, because the conceptus is transparent, cell-cell associations can be studied until well after the hypoblast is formed. These combined features mean that lineage allocation and axis formation can be more easily studied and related to oocyte/zygote polarity and the order of cell division in these conceptuses. Another feature is the ability to collect normal unattached embryonic and fetal stages in some species and to culture conceptuses from the primitive streak stage to within a few hours of birth. This ability means that stages of organogenesis in most organ systems are readily accessible without the complications of implantation.

Dystroglycan down-regulation links EGF receptor signaling and anterior–posterior polarity formation in the Drosophila oocyte

Anterior-posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. Here, we show that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. Our data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior-posterior polarity formation in the oocyte.

Reproductive Structure of the Adult Green Sturgeon, Acipenser medirostris, Population in the Rogue River, Oregon

The primary objective of this study was to determine the reproductive structure of the adult green sturgeon population in the Rogue River. Green sturgeon were captured by gillnet in the lower 11.6-68.4 river kilometers in April to July 2000-2003 and September and October 2002-2003. Gonadal tissue, collected by biopsy, was processed histologically, blood was collected from the caudal vasculature, and fork length (FL) and total length (TL) (±0.5 cm) were measured for each individual. Sex steroids, testosterone (T), 11-ketotestosterone (11-KT), and estradiol-17b (E2), were measured by radioimmunoassay. Bio- logical samples were collected from a total of 88 green sturgeon of which 37 females and 41 males were confirmed by histological analysis. Four gravid females, captured in the spring, were visually identified, and oocyte polarization index and ovarian follicle diameter indicated that these females were in spawning condition. Gonadal samples collected from six individuals did not contain gonial cells, hence the sex and stage of maturity in these individuals remains unknown. Of the 20 females captured in the spring, 1 was vitellogenic, 4 were post-vitellogenic, and 15 were post-ovulatory. Twenty-one females were cap- tured in the fall of which 6 were pre-vitellogenic, 7 vitellogenic, and 8 post-ovulatory. Of the 16 males captured in the spring, 2 were pre-meiotic, 8 were ripe or actively spermiating, and 6 were post-spermiation. Twenty-five males were cap- tured in the fall: 11 pre-meiotic males and 14 post- spermiation. The majority of green sturgeon captured in the Rogue River were reproductively active or had recently spawned indicating the importance of this river for the preservation of green sturgeon.

Drosophila Anterior-Posterior Polarity Requires Actin-Dependent PAR-1 Recruitment to the Oocyte Posterior

The Drosophila anterior-posterior axis is established at stage 7 of oogenesis when the posterior follicle cells signal to polarize the oocyte microtubule cytoskeleton. This requires the conserved PAR-1 kinase, which can be detected at the posterior of the oocyte in immunostainings from stage 9. However, this localization depends on Oskar localization, which requires the earlier PAR-1-dependent microtubule reorganization, indicating that Oskar-associated PAR-1 cannot establish oocyte polarity. Here we analyze the function of the different PAR-1 isoforms and find that only PAR-1 N1 isoforms can completely rescue the oocyte polarity phenotype. Furthermore, PAR-1 N1 is recruited to the posterior cortex of the oocyte at stage 7 in response to the polarizing follicle cell signal, and this requires actin, but not microtubules. This suggests that posterior PAR-1 N1 polarizes the microtubule cytoskeleton. PAR-1 N1 localization is mediated by a cortical targeting domain and a conserved anterior-lateral exclusion signal in its C-terminal linker domain. PAR-1 is also required for the polarization of the C. elegans zygote and is recruited to the posterior cortex in an actin-dependent manner. Our results therefore identify a molecular parallel between axis formation in Drosophila and C. elegans and make Drosophila PAR-1 N1 the earliest known marker for the polarization of the oocyte.

Drosophila Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis

The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. We have determined that the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, “dumping” of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis.

Space Asymmetry Directs Preferential Sperm Entry in the Absence of Polarity in the Mouse Oocyte

Knowledge about the mechanism that establishes embryonic polarity is fundamental in understanding mammalian development. In re-addressing several controversial claims, we recently proposed a model in which mouse embryonic polarity is not specified until the blastocyst stage. Before fertilization, the fully differentiated oocyte has been characterized as “polarized,” and we indeed observed that the sperm preferentially enters the polar body half. Here we show that preferential sperm entry is not due to an intrinsic polarity of the oocyte, since fertilization takes place uniformly when the zona pellucida is removed. We suggest that the term “asymmetry” denotes morphological differences, whereas “polarity” in addition implies developmental consequences. Thus, the mouse oocyte can be considered “asymmetric” but “non-polarized.” The penetration through the zona pellucida is also random, and a significant proportion of sperm binds to the oocyte membrane at a point distant from the zona penetration site. Time-lapse recordings confirmed that sperm swim around the perivitelline space before fertilization. Experimental enlargement of the perivitelline space in the non-polar body half increased the regional probability of fertilization. Based on these experiments, we propose a model in which the space asymmetry exerted by the first polar body and the zona pellucida directs sperm entry preferentially to the polar body half, with no need for oocyte polarity.

Regulation of dynamic events by microfilaments during oocyte maturation and fertilization

Actin filaments (microfilaments) regulate various dynamic events during oocyte meiotic maturation and fertilization. In most species, microfilaments are not required for germinal vesicle breakdown and meiotic spindle formation, but they mediate peripheral nucleus (chromosome) migration, cortical spindle anchorage, homologous chromosome separation, cortex development/maintenance, polarity establishment, and first polar body emission during oocyte maturation. Peripheral cortical granule migration is controlled by microfilaments, while mitochondria movement is mediated by microtubules. During fertilization, microfilaments are involved in sperm incorporation, spindle rotation (mouse), cortical granule exocytosis, second polar body emission and cleavage ring formation, but are not required for pronuclear apposition (except for the mouse). Many of the events are driven by the dynamic interactions between myosin and actin filaments whose polymerization is regulated by RhoA, Cdc42, Arp2/3 and other signaling molecules. Studies have also shown that oocyte cortex organization and polarity formation mediated by actin filaments are regulated by mitogen-activated protein kinase, myosin light-chain kinase, protein kinase C and its substrate p-MARKS as well as PAR proteins. The completion of several dynamic events, including homologous chromosome separation, spindle anchorage, spindle rotation, vesicle organelle transport and pronuclear apposition (mouse), requires interactions between microfilaments and microtubules, but determination of how the two systems of the cytoskeleton precisely cross-link, and which proteins link microfilaments to microtubules to perform functions in eggs, requires further studies. Finally, the meaning of microfilament-mediated oocyte polarity versus embryo polarity and embryo development in different species (Drosophila, Xenopus and mouse) is discussed.

The centrosome-nucleus complex and microtubule organization in theDrosophilaoocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. We have identified a centriole-containing centrosome close to the oocyte nucleus. Remarkably, the centrosomal components, gamma-tubulin and Drosophila pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. We further report that the MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. We propose that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization.

The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes (gamma-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical "organizing pole" is hypothesized.

Direct association of Bazooka/PAR-3 with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositide signaling

Cell polarity in Drosophila epithelia, oocytes and neuroblasts is controlled by the evolutionarily conserved PAR/aPKC complex, which consists of the serine-threonine protein kinase aPKC and the PDZ-domain proteins Bazooka (Baz) and PAR-6. The PAR/aPKC complex is required for the separation of apical and basolateral plasma membrane domains, for the asymmetric localization of cell fate determinants and for the proper orientation of the mitotic spindle. How the complex exerts these different functions is not known. We show that the lipid phosphatase PTEN directly binds to Baz in vitro and in vivo, and colocalizes with Baz in the apical cortex of epithelia and neuroblasts. PTEN is an important regulator of phosphoinositide turnover that antagonizes the activity of PI3-kinase. We show that Pten mutant ovaries and embryos lacking maternal and zygotic Pten function display phenotypes consistent with a function for PTEN in the organization of the actin cytoskeleton. In freshly laid eggs, the germ plasm determinants oskar mRNA and Vasa are not localized properly to the posterior cytocortex and pole cells do not form. In addition, the actin-dependent posterior movement of nuclei during early cleavage divisions does not occur and the synchrony of nuclear divisions at syncytial blastoderm stages is lost. Pten mutant embryos also show severe defects during cellularization. Our data provide evidence for a link between the PAR/aPKC complex, the actin cytoskeleton and PI3-kinase signaling mediated by PTEN.

Cell polarity during folliculogenesis and oogenesis

Polarity is an important aspect of oogenesis and early development for many animal groups, but only recently it has become relevant to the study of mammals. Mammalian oocyte development occurs through tight coordination and interaction between all ovarian structures. In fact, bi-directional communication between the oocyte and its companion granulosa cells (GC) in the ovarian follicle seems essential for GC proliferation, differentiation, and production of a functional female gamete. The trans-zonal projections (TZP), which are specialized extensions from granulosa cells that terminate on the oolema after crossing the zona pellucida, are major structural components necessary for oocyte–GC interaction. Granulosa cell polarity seems to be a necessary requisite for appropriate function of TZP, and the role of FSH as modulator of a polarized phenotype on GC is discussed. This article also discusses oocyte polarity with special reference to the partial loss of polarity that occurs during in-vitro oocyte maturation and possible implications in the modulation of oocyte competencies. Cytoskeletal markers that may account for oocyte quality were defined and found to be distinct in in-vivo and in-vitro matured oocytes. Implications of partial loss of oocyte polarity during in-vitro maturation, reflected by distinct distribution of these markers, are further discussed. It is also proposed that expression of both somatic and germ cell polarity in the ovarian follicle will ultimately determine acquisition of meiotic, fertilization and developmental competences by the oocyte.

The developmental origins of mammalian oocyte polarity.

Drosophila has been an excellent model system to study the cell and molecular determinants of oocyte axis specification, a problem which is little known in mammalian species. Recent evidence supports the notion that mammalian oocytes utilize axis-orienting properties during the course of oogenesis. Among these, axis specification in relation to the oocyte cortex, germinal vesicle (GV) position, anchoring of GV and spindle, and patterning of follicle cell/oocyte attachments are proposed as conserved features of oogenesis in mammals that may be important to the survival and development of the preimplantation embryo.

New Synaptic Bouton Formation Is Disrupted by Misregulation of Microtubule Stability in aPKC Mutants

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. Here we show that at Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1Brelated protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, we show that Baz and Par-6 are also expressed at synapses and that their synaptic localization depends on aPKC activity. Our findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory.

Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate.

In many cell types polarized transport directs the movement of mRNAs and proteins from their site of synthesis to their site of action, thus conferring cell polarity. The cytoplasmic dynein microtubule motor complex is involved in this process. In Drosophila melanogaster, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for the transport of macromolecules to the oocyte and to the apical surface of the blastoderm embryo. Hence, Egl and BicD, which have been shown to associate, may be part of a conserved core localization machinery in Drosophila, although a direct association between these molecules and the dynein motor complex has not been shown. Here we report that Egl interacts directly with Drosophila dynein light chain (Dlc), a microtubule motor component, through an Egl domain distinct from that which binds BicD. We propose that the Egl-BicD complex is loaded through Dlc onto the dynein motor complex thereby facilitating transport of cargo. Consistent with this model, point mutations that specifically disrupt Egl-Dlc association also disrupt microtubule-dependant trafficking both to and within the oocyte, resulting in a loss of oocyte fate maintenance and polarity. Our data provide a direct link between a molecule necessary for oocyte specification and the microtubule motor complex, and supports the hypothesis that microtubule-mediated transport is important for preserving oocyte fate.

Two PAR6 Proteins Become Asymmetrically Localized during Establishment of Polarity in Mouse Oocytes

Meiotic maturation in mammals is characterized by two asymmetric divisions, leading to the formation of two polar bodies and the female gamete. Whereas the mouse oocyte is a polarized cell, molecules implicated in the establishment of this polarity are still unknown. PAR proteins have been demonstrated to play an important role in cell polarity in many cell types, where they control spindle positioning and asymmetric distribution of determinants. Here we show that two PAR6-related proteins have distinct polarized distributions in mouse oocytes. mPARD6a is first localized on the spindle and then accumulates at the pole nearest the cortex during spindle migration. In the absence of microtubules, the chromosomes still migrate to the cortex, and mPARD6a was found associated with the chromosomes and was facing the cortex. mPARD6a is the first identified protein to associate with the spindle during spindle migration and to relocalize to the chromosomes in the absence of microtubule behavior, suggesting a role in spindle migration. The other protein, mPARD6b, was found on spindle microtubules until entry into meiosis II and relocalized to the cortex at the animal pole during metaphase II arrest. mPARD6b is the first identified protein to localize to the animal pole of the mouse oocyte and likely contributes to the polarization of the cortex.

Abstrakt, a DEAD Box Protein, Regulates Insc Levels and Asymmetric Division of Neural and Mesodermal Progenitors

Asymmetric cell division generates cell diversity in bacteria, yeast, and higher eukaryotes [1–3]. In Drosophila, both neural and muscle progenitors divide asymmetrically [4–16]. In these cells the Inscuteable (Insc) protein complex coordinates cell polarity and spindle orientation. Abstrakt (Abs) is a DEAD-box protein that regulates aspects of cell polarity in oocytes and embryos [17]. We use a conditional allele of abs to investigate its role in neural and muscle progenitor cell polarity. In neuroblasts we observe loss of apical Insc crescents, failure in basal protein targeting, and defects in spindle orientation. In the GMC4-2a cell we observe loss of apical Insc crescents, defects in basal protein targeting, and equalization of sibling neuron fates; muscle precursors show a similar equalization of sibling cell fates. These phenotypes resemble those of insc mutants; indeed, abs mutants show a striking loss of Insc protein levels but no change of insc RNA levels. Furthermore, we find that the Abs protein physically interacts with insc RNA. Our results demonstrate a novel role for Abs in the posttranscriptional regulation of insc expression, which is essential for proper cell polarity, spindle orientation, and the establishment of distinct sibling cell fates within embryonic neural and muscle progenitors.