Because shot RNA is more broadly expressed than the protein, this spatially restricted pattern seems to be achieved by restricting where shot RNA is translated and by limiting the diffusion of Shot protein through its interactions with the actin cytoskeleton in anterior regions Although no deficits in the actin cytoskeleton were detected in shot mutant oocytes, fewer microtubules occupied anterior regions of later-stage oocytes, indicating that Shot promotes regional microtubule assembly or locally stabilizes existing microtubules 34 , Consistent with this model, the actin-binding domain of Shot is required for anchoring of non-centrosomal microtubule-organizing centers MTOCs , and defective anchoring of acentrosomal MTOCs, as occurs in shot mutants, leads to microtubule disorganization 34 , Like Patronin, Katanin, a microtubule-severing protein, localizes to the anterior cortex 34 Figure 1A.
Taken together, these recent studies provide evidence for a spatially restricted, actin-tethered system to generate MTOCs independent of centrosomes. It will be interesting to determine whether Spektraplakins contribute to meiotic spindle organization or position during meiotic divisions as a component of, or in a manner analogous to, the actin meshwork of mouse and starfish oocytes discussed above. Therefore, actin binding may be required indirectly or directly for Mgn association with the vegetal cortex, since Balbiani body cargo are not delivered in mgn mutants predicted to encode for proteins that partially disrupt the actin-binding domain 36 Figure 1B, C.
However, based on epistasis analysis, it appears that concurrent loss of buc and magellan produces additive phenotypes, oocytes with acentric nuclei and lacking a Balbiani body core marked by Buc protein and densely populated mitochondria Therefore, Macf1 regulates nuclear position by a mechanism that is independent of impaired Balbiani body dynamics.
Whether detachment of microtubules from the cortex, as occurs in magellan mutants lacking the microtubule-binding domain, still occurs in the actin-binding deletion alleles or in the absence of Buc and the Balbiani body has yet to be reported. The molecular factors and relevant interactions remain to be determined. The importance of chromosomal cues to the organization of the meiotic spindle in oocytes, which, unlike mitotic cells, lack centrosomes and tend to be large cells, has been appreciated for more than a decade.
In the absence of centrosomes, the chromosomes emerged as a potential catalyst of spindle assembly by a mechanism thought to involve microtubule nucleation or capture. Three pathways to organize the spindle without centrosomes have been defined: two involving chromatin capture and a third that is thought to bias elongation toward the poles. Consistent with this activity, interference with Augmin in Xenopus and Drosophila diminishes nucleation of microtubules associated with the spindle 44 , The observation that Augmin complex components are enriched at the poles in Drosophila oocytes, but not S2 cultured Drosophila somatic cells , and that Augmin components turn over more slowly based on fluorescence recovery after photobleaching FRAP experiments has led to a model whereby in oocytes Augmin activity is biased toward the poles The third pathway, the CPC pathway, stabilizes microtubules and promotes spindle assembly in Xenopus egg extracts and in zebrafish and Drosophila oocytes and early embryos 46 — Although the CPC, a key complex in spindle morphogenesis, and in particular the kinase subunit Aurora A, which associates with chromatin and is essential for spindle I assembly, was implicated as a component of a chromosome-based cue system 48 , 51 , 53 , 54 , it was not clear how such spatial information would be transmitted from the chromosomes to promote spindle organization or dynamics.
In addition, it was not clear how independently each of these organizing systems operates within cells or species. Indeed, interference with the Ran pathway in Drosophila and mouse oocytes did not abolish assembly of the meiosis I spindle, and although complete loss of CPC function blocks spindle microtubules altogether, partial loss of function significantly delays assembly 48 , 51 , 55 , 56 , indicating that some of these mechanisms may be redundant or that additional unknown mechanisms are deployed in cells lacking centrosomes.
Similarly, depletion of two plus end-directed microtubule-associated kinesin motors, subito sub or kinesin 6 , and non-claret disjunctional ncd , first observed in and later shown to be a Kinesin 14, disrupts spindle morphogenesis 58 — In the case of ncd mutants, spindle polarity defects were associated with failure to cluster microtubule minus ends and failure to localize Msps to the spindle poles 57 , 61 , Based on the observation that the localization of Msps, but not D-TACC drosophila transforming, acidic, coiled-coil containing , another spindle pole and centrosome-localized protein, was disrupted in ncd mutants, a model was put forth wherein the minus end-directed motor Ncd transports Msps to the spindle pole where it interacts with and is anchored by its binding partner, D-TACC, which localizes earlier by a mechanism that does not require Ncd Moreover, based on the observations that d-tacc mutant oocytes have similar spindle defects and that Msps localization requires D-TACC, interaction between these two proteins specifically at the spindle pole could then stabilize the bipolar meiotic spindle Based on its localization and activity in microtubule assembly assays in other organisms, Ran was postulated to be a candidate factor emanating from the chromosomes to set up a gradient capable of triggering spindle assembly 67 , 68 ; however, among the components examined, only CPC was shown to be required for meiosis I spindle formation in Drosophila 48 , 51 , 55 , Therefore, at least two key questions remained to be addressed: what is the identity of the putative factor that establishes the spindle-organizing region and promotes chromatin-mediated spindle assembly, and what is responsible for organizing the bipolar spindle in the absence of centrosomes?
Unique structural features and activities of individual Kinesin classes and studies of mitotic cells and in vitro assays indicated that the problem of spindle organization was more interesting and complicated than simple motor-mediated transport along microtubules 69 — Antagonistic actions of Kinesins had been observed in Drosophila , mammalian, and yeast cells 74 — A few years later, in vitro assays using fluorescently labeled microtubules and tagged Kinesin 5 Eg 5 , long suspected of being involved in microtubule manipulation because of its unique bipolar structure with motors at both ends of the stalk, showed that Kinesin 5 could slide microtubules in an orientation-dependent manner Similarly, photoactivation of a light-inducible Kinesin5paGFP and photobleaching of labeled microtubules obtained from Xenopus egg extracts provided evidence that both Kinesin 5 and microtubules were mobile in the middle of the spindle and that Kinesin 5 movement toward and concentration at the spindle poles was Dynein-dependent, as p could block this Together, these studies indicated that Kinesin 5 slides parallel microtubules but locks anti-parallel microtubules, providing evidence for a model wherein opposing motors could supply unique activities during spindle morphogenesis.
For anti-parallel microtubules, the forces associated with transport cause the microtubules to slide in opposite directions, but when the microtubules are oriented the same way, switching of the motors between microtubules has been proposed to generate opposing forces that effectively lock the microtubules in place 79 Figure 2A. Cumulatively, these and other studies of Kinesin activities in mitotic cells and in vitro systems provided support for models in which selective sorting and entrapment of spindle microtubules could be accomplished by balanced but opposing forces produced by distinct molecular motors.
Moreover, it opened up questions about whether localized and selective activity of molecular motors might contribute to meiotic spindle assembly and, if so, how such activities would be regulated, particularly in cells lacking centrosomes, such as oocytes. A Illustrations depicting how Kinesins can crosslink microtubules to promote the sliding and clustering necessary for sorting of anti-parallel microtubules and spindle assembly and stability. B Drawing depicts the Ran-Importin pathway described for Xenopus egg extracts. Kinesin 14 interacts with Importin and is inhibited from interacting with microtubules.
Importin inhibition is alleviated near the chromosomes, where Ran-GTP concentration is high. Ran-GTP association with Importin allows Kinesin 14 to associate with kinetochore-bound microtubules. Balancing forces from Kinesin 5 stabilize the bipolar spindle.
Gametogenesis in Humans
C Illustration of a pathway for meiotic spindle assembly in Drosophila oocytes. Kinesin 14 interacts with and is inhibited from interacting with non-spindle microtubules. Near the chromosomes, Aurora B phosphorylation dissociates and unmasks the repressed microtubule-organizing activity of Kinesin As in Xenopus extracts, balancing forces from Kinesin 5 and two additional Kinesins promote assembly and stabilization of the bipolar spindle. The functions of spindle proteins that are required specifically in meiotic cells, either because these genes are expressed only in meiotic cells or because the genes are expressed in and localized to the spindle poles of both mitotic and meiotic cells but act redundantly with other mitotic spindle components, can be studied with traditional mutagenesis approaches as long as they are viable to reproductive stages.
In contrast to those proteins, Kinesin 5 Klp61F in Drosophila localizes to centrosomal and acentrosomal spindle poles in Drosophila and mammalian cells 80 — In mitotic cells, Kinesin 5 prevents the centrosomes from collapsing 77 , and thus its functions in meiotic spindle morphogenesis could not be determined. As in mitotic cells, interference with Kinesin 5 using the inhibitor monastral caused a monopolar meiotic spindle phenotype in mammalian oocytes 81 , Further highlighting the complexity of meiotic spindle morphogenesis, when similar depletion assays were used, aspects of the meiotic spindle defects were shown to depend on another kinesin, Kinesin 12, and the microcephaly-associated protein, ASP In that context, cell culture-based activity assays demonstrated that Asp bundles minus ends to other microtubules within the spindle, and at the spindle pole, and is thought to act by an Ncd-independent mechanism based on the intact localization of Asp-GFP to mitotic spindles of Ncd RNA interference RNAi -depleted S2 cells and failure of Asp and Ncd to compensate for one another in depletion and rescue studies in cultured Drosophila somatic cells This differential effect may be due to technical differences; however, because these distinct functions are observed within the same organism, they cannot be attributed simply to species differences.
Instead, it may reflect differences between meiotic and mitotic spindle composition, including obviously the absence of centrosomes in oocytes. Taken together, these comparisons suggest that differential use of molecular motors seems to allow the meiotic spindle of oocytes to supply forces or activities that are supplied by the centrosome in mitotic cells.
How these activities are spatially and temporally restricted remains an open question. Among the molecules that they bind to and regulate are several key signaling proteins, including kinases involved in cell cycle control 88 — The lack of intrinsic enzymatic activity and identifiable targeting motifs led to models of action whereby binding interferes with any functional activity proximal to the interaction domain of the target protein Because of their mode of action and roles in signal transduction and cell cycle, proteins are compelling candidates for involvement in oogenesis.
Indeed, proteins have been shown to play important roles in spindle morphogenesis in mice and Drosophila 94 , In Drosophila , another protein promotes MTOC formation in egg chambers and is required for oocyte specification Evidence for deficits in MTOC formation include deficits in the localization of mini spindles, a microtubule-associated protein that normally localizes to sites of microtubule nucleation In this context, proteins are thought to promote selective interaction between Ncd Kinesin 14 and spindle microtubules while preventing Ncd association with non-spindle microtubules This new mechanism appears to share features with the well-characterized Ran-Importin system used in Xenopus 40 Figure 2B, C.
Specifically, blocks Kinesin 14 interaction with non-spindle microtubules in a manner that is analogous to Importin Figure 2B, C.
As mentioned above, in the mouse, a different protein has been implicated in spindle morphogenesis Nonetheless, local regulation of the activity of molecular motors with microtubule bundling functions seems to play a conserved role in generating opposing forces to provide for robust spindle assembly in the absence of centrosomes and possibly to provide insurance for equal chromosome segregation between the large oocyte and tiny polar bodies produced from meiotic division.
Production of a developmentally competent oocyte is essential for normal development of an individual and survival of species. The oocyte is a highly specialized and enormous cell that must retain the capacity to give rise to all of the cells that make up an embryo. Thus, compared with somatic cells, the oocyte has unique challenges, including accomplishing an asymmetric division that directs the bulk of the maternal cytoplasm to the oocyte but equally distributes the chromosomes in the absence of centrosome-based MTOCs. The recent evidence discussed in this review indicates that assembly of the meiotic spindle is orchestrated via mechanisms that involve spatially restricted cues, including factors emanating from the chromosomes, that allow kinesin motors with microtubule-organizing activity to act only on subsets of microtubule to establish balanced action of motors with opposing activities, thus substituting for functions supplied by the centrosome in mitotic cells.
Coordination between cytoskeletal elements, in part through the activity of crosslinking proteins, impacts cellular and meiotic spindle morphology to ensure that the meiotic divisions and oogenesis are successful. As discussed, several mechanisms to support microtubule nucleation and spindle morphogenesis have been discovered. Much of what we understand has come from basic genetics, including targeted and forward genetic screens, and pharmacological approaches.
As highlighted herein, improved genome editing and reverse genetics tools coupled with elegant in vivo labeling and imaging approaches have already shed significant light on this process and will continue to do so. These technological advances and those to follow will make it feasible to systematically test candidate factors to decipher their contribution to assembly of the meiotic spindle and chromosome segregation.
This is a significant biological problem with clear potential to impact reproduction and fertility, as most first-trimester miscarriages in humans are associated with defects in chromosome segregation and aneuploidy.
No competing interests were disclosed. Research on the germ line, oocyte polarity, and fertility in the Marlow lab is supported by NIH R01 GM and start-up funds to the author. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. F Faculty Reviews are written by members of the prestigious F Faculty. They are commissioned and are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. The reviewers who approved the final version are listed with their names and affiliations.
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Sign up for content alerts and receive a weekly or monthly email with all newly published articles. Register with FResearch. Already registered? Sign in. In this review, we will summarize the effects of altering the synthesis of N-glycans, O-glycans, proteoglycans, glycophosphatidylinositol GPI anchored proteins, and glycolipids during gametogenesis in the mouse.
Glycosylation genes whose deletion causes embryonic lethality have been investigated following conditional deletion using various Cre recombinase transgenes with a cell-type specific promoter.
Oogenesis - 1st Edition
The potential effects of mutations in corresponding glycosylation genes of humans will be discussed in relation to consequences to fertility and potential for use in contraception. The mammalian glycome is defined by the genes that encode activities required for the synthesis of glycosylated proteins and lipids. These include genes that encode glycosyltransferases, nucleotide sugar synthases, nucleotide sugar modifiers, nucleotide sugar transporters and glycosidases required to prune glycans during synthesis. Also required for optimal glycosylation are proteins that maintain the structure and environment of the secretory pathway.
A summary of the classes of glycan synthesized by mammals is given in Figure 1. The actual complement of glycan structures expressed in the endoplasmic reticulum ER , in Golgi compartments, at the cell surface, or secreted by a mammalian cell will depend on the glycosylation-related genes that are active in that cell, and that spectrum will likely vary at different stages of development or differentiation. With few exceptions, all glycoproteins, proteoglycans and glycolipids are synthesized in the secretory pathway, although N-glycans and glycosylphosphatidylinositol GPI anchors begin their synthesis on the cytoplasmic side of the ER membrane, before an immature glycan is flipped to the luminal side.
Maturation continues in the ER and Golgi compartments. Thus, effects on the structure or biochemical environment of the ER and Golgi compartments may affect glycosylation and alter the nature of the glycans produced. The glycans on mature glycoproteins and glycolipids expressed at the cell surface or secreted from a cell are the functional glycans involved in recognition by glycan binding proteins on other cells, and in the extracellular matrices of a tissue. Glycoprotein exceptions to synthesis in the secretory pathway are proteins modified on Ser or Thr by O-GlcNAc in the cytoplasm or nucleus Zachara et al.
Figure 1. Cell Surface Glycans in Mammals. The diagram depicts one or more glycans from each class of mammalian glycan. The diagram is modified from Figure in Stanley with permission. Sugar symbols are according to the Symbol Nomenclature for Glycans Varki et al. A commonly used strategy to determine if a particular glycan or sugar residue is necessary for the development or differentiation of a specific cell type is to inactivate or delete a glycosylation gene.
However, this has the drawback that many glycosylated molecules of different functions will be altered. To address functions for glycans in individual glycoproteins, one or more known glycosylation sites can be mutated to preclude their modification. However, relatively few glycans are added at a known amino acid, making this approach arduous, although useful once glycosylation sites have been established. This review will describe the consequences for oogenesis Figure 2 and spermatogenesis Figure 3 of altering glycan synthesis by targeted inactivation of glycosylation genes responsible for the synthesis of Golgi glycans in the mouse.
http://john-und.sandra-gaertner.de/aprendiendo-ingls-como-un-nio.php These glycans are identified in Figures 1 , 4. Until recently, glycans in the ovary and testis have been investigated by immunohistochemistry and other histochemical assays and by using glycan binding proteins such as plant lectins Lee and Damjanov, ; Wu et al. However, the application of matrix-assisted laser desorption mass spectrometry imaging MALDI-MSI has begun to reveal the molecular nature of glycans along with their location in complex tissues Drake et al.
Figure 2. Oogenesis in Mammals. The diagram describes the different stages of oogenesis and identifies the follicular stage in which the ZP3 and Vasa promoters are active in oocytes.