Centrosome dynamics during the meiotic progression in the mouse oocyte (original) (raw)
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Centrosome and microtubule dynamics during early stages of meiosis in mouse oocytes
Molecular Human Reproduction, 2003
Centrosomes, major regulatory sites for the microtubule (MT) nucleation, are regulated in a dynamic manner throughout the process of meiotic maturation. Recently, centrosome orientation in mouse oocytes has been demonstrated in metaphase I through metaphase II. However, centrosomal protein expression in concordance with MT polymerization in earlier stages of oocyte maturation from germinal vesicle stage (GV) to prometaphase I still remains unclear. The present study aims to assess the centrosome±microtubule remodelling during the onset of meiosis based on strict criteria of nuclear maturation. Six consecutive stages were determined for scoring the oocytes as unrimmed nucleolus (UR), partially rimmed nucleolus (PR), fully rimmed nucleolus (FR), nuclear lamina dissolution (NLD), disappearance of nucleolus (DON), and chromatin condensation (CC). A centrosomal protein, pericentrin, was found tightly localized adjacent to nuclear lamina in UR, lacking any MT nucleation activity. In concordance with the competency to resume meiosis, an increase in the amount and nucleation capacity of pericentrin is noted. In FR, cytoplasmic MT almost disappeared while de-novo microtubule polymerization was found in small aggregates of pericentrin localized around the nucleus. Towards the end of DON and CC, a sudden burst of pericentrin was noted with an extreme MT nucleation activity in an organized fashion that is essential for the rapid formation of ®rst meiotic spindle. The results show that centrosomes display precisely controlled spatio-temporal changes during the onset of meiotic maturation. Accumulation of centrosomal proteins to a single locus followed by a sequestration to several spots might be evidence of a mechanism by which the proper distribution of centrosomal material during nuclear breakdown and subsequently formation of spindle are regulated in concordance with the nuclear maturation.
Journal of Cell Biology, 1992
Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native cent...
Proceedings of the …, 1986
The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immunofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and condense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitution of the daughter nuclei, the centrosomal material is dposed as hemispherical caps around the poleward surfaces
Journal of Cell Biology, 1984
We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injectio...
The Journal of cell biology, 1984
We report the results of studies in which partially purified centrosomes, nuclei, and DNA were injected into frog's eggs, which are naturally arrested in metaphase or interphase. These results have led to an independent assessment of the contributions of the centrosome and the chromatin to the formation of the mitotic spindle and suggest a simple explanation for the transition from interphase to metaphase microtubule arrays.
Differential regulation of maternal vs. paternal centrosomes
Proceedings of the National Academy of Sciences, 1999
Centrosomes are the main microtubuleorganizing centers in animal cells. During meiosis and mitosis, two centrosomes form the poles that direct the assembly of a bipolar spindle, thus ensuring the accurate segregation of chromosomes. Cells cannot tolerate the presence of more than two active centrosomes during meiosis or mitosis because doing so results in the formation of multipolar spindles, infidelity in chromosome segregation, and aneuploidy. Here, we show that fertilization of Spisula solidissima oocytes results in cells that contain three active centrosomes, two maternal and one paternal. During meiosis I, the paternal centrosome's ability to nucleate microtubules is selectively shut off while maternal centrosomes remain competent to nucleate microtubules and assemble asters in the same cytoplasm. We propose that embryos can identify paternal vs. maternal centrosomes and can control them differentially.
Centrosome biogenesis continues in the absence of microtubules during prolonged S-phase arrest
Journal of Cellular Physiology, 2010
When CHO cells are arrested in S-phase, they undergo repeated rounds of centrosome duplication without cell cycle progression. While the increase is slow and asynchronous, the number of centrosomes in these cells does rise with time. To investigate mechanisms controlling this duplication, we have arrested CHO cells in S-phase for up to 72 hours, and coordinately inhibited new centriole formation by treatment with the microtubule poison colcemid. We find that in such cells, the pre-existing centrosomes remain, and a variable number of foci -containing α/γ-tubulin and centrin 2 -assemble at the nuclear periphery. When the colcemid is washed out, the nuclearassociated foci disappear, and cells assemble new centriole-containing centrosomes, which accumulate the centriole scaffold protein SAS-6, nucleate microtubule asters, and form functional mitotic spindle poles. The number of centrosomes that assemble following colcemid washout increases with duration of S-phase arrest, even though the number of nuclear-associated foci or pre-existing centrosomes does not increase. This suggests that during S-phase, a cryptic generative event occurs repeatedly, even in the absence of new triplet microtubule assembly. When triplet microtubule assembly is restored, these cryptic generative events become realized, and multiple centriole-containing centrosomes assemble.
1988
Centrosomes undergo cell cycle-dependent changes in shape and separations, changes that govern the organization of the cytoskeleton. The cytoskeleton is largely organized by the centrosome; however, this investigation explores the importance of cytoskeletal elements in directing centrosome shape. Since the sea urchin egg during fertilization and mitosis displays dramatic and synchronous changes in centrosome shape, the effects of cytoskeletal inhibitors on centrosome compaction, expansion, and separation were explored by the use of anticentrosome immunofluorescence microscopy. Centrosome expansion and separation was studied during two phases: the transition after sperm incorporation, when the compact sperm centrosome enlarges and the sperm aster develops, and from prometaphase to telophase, when the compact spindle poles enlarge. Compaction was investigated when the dispersed centrosome at interphase condenses into the two spindle poles at prometaphase. Although centrosome expansion and separation typically occur concurrently, P-mercaptoethanol results in centrosome separation independent of expansion. Microtubule inhibitors prevent centrosome expansion and separation, and expanded centrosomes collapse. Since pronuclear union is arrested by microtubule inhibitors, this treatment also affords the opportunity to explore the relative attractiveness of the male and female pronuclei for these centrosomal antigens. Both pronuclei acquire centrosomal material; though only the male centrosome is capable of organizing a functional bipolar mitotic apparatus at first division, the female centrosome nucleates a monaster. Microfilament inhibition (cytochalasin D) prevents centrosome separation but not expansion or compaction. These results demonstrate that as the centrosome shapes the cytoskeleton, the cytoskeleton alters centrosome shape.