The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome recruitment

The gamma-tubulin ring complex (gammaTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that gammaTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the gammaTuRC. Consequently, the cap structure of the gammaTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. Here, we report the cloning and characterization of a new subunit of Xenopus gammaTuRC, Xgrip210. We show that Xgrip210 is a conserved centrosomal protein that is essential for the formation of gammaTuRC. Using immunogold labeling, we found that Xgrip210 is localized to the ends of microtubules nucleated by the gammaTuRC and that its localization is more distal, toward the tip of the gammaTuRC-cap structure, than that of gamma-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the gammaTuRC, but also the recruitment of gamma-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the gammaTuRC cap structure that is required for the assembly of the gammaTuRC.     

Effects of RNase and RNA on in vitro aster assembly

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polucytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing condition. These results demonstrate that specfic polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.     

A potential role for human cohesin in mitotic spindle aster assembly

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to form in vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.     

Chromatin assembly in Xenopus oocytes: In vitro studies

We describe and characterize a complex reaction that catalyzes DNA supercoiling and chromatin assembly in vitro. A Xenopus oocyte extract supplemented with ATP and Mg⁺⁺ converts DNA circles into minichromosomes that display a native, 200 bp periodicity. When supercoiled DNA is added to this extract it undergoes a time-dependent series of topological changes, which precisely mimic those found when the DNA is microinjected into oocytes. As judged by the conformation of the subsequently deproteinized DNA, the supercoiled DNA is first relaxed, in a reaction that takes 4 min, and then it is resupercoiled in a slower process that takes 4 hr. The relaxation is partially inhibited by EDTA, to an extent that suggests that that it is catalyzed by a type I DNA topoisomerase. The resupercoiling, on the other hand, requires ATP and Mg⁺⁺, is completely inhibited by EDTA, and is inhibited by novobiocin in a manner that suggests it is catalyzed by a type II DNA topoisomerase. These findings, and the ones reported in the preceding paper (Ryoji and Worcel, 1984), lead us to propose that chromatin assembly is an active, ATP-driven process.     

Centrosome reduction during Rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration

Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.     

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.     

Attachment of boar sperm to mucosal explants of oviduct in vitro: possible role in formation of a sperm reservoir

Ejaculated boar sperm were incubated with explants of porcine oviductal mucosa that had been dissected from the isthmic and ampullar regions of gilts. Sperm bound within minutes to the epithelial surfaces of the explants. Binding was not affected by region (isthmus or ampulla) nor day of estrous cycle (Day 0 or Day 10), but was increased by addition of 70 pg/ml 17 beta-estradiol to the medium. Scanning electron micrographs indicated that sperm bound, via the acrosomal region, to ciliated cells. After 24 h, the numbers of bound sperm dropped significantly, but the motility of the bound sperm did not. A mucous material that entrapped sperm was observed on the epithelial surfaces of 23/32 isthmic and only 4/32 ampullar explants. These results indicate that sperm sticking to ciliated cells and mucus can create a sperm reservoir in the isthmus, but the means by which sperm are released remain unknown.     

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.     

Nuclear assembly of demembranated Xenopus sperm in plant cell-free extracts from Nicotiana ovules

The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei. Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X. leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation. Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin. Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion. After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each.     

Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly

We find that the remodeling of the condensed Xenopus laevis sperm nucleus into the paternal pronucleus in egg extracts is associated with phosphorylation of the core histones H2A, H2A.X and H4, and uptake of a linker histone B4 and a HMG 2 protein. Histone B4 is required for the assembly of chromatosome structures in the pronucleus. However neither B4 nor core histone phosphorylation are required for the assembly of spaced nucleosomal arrays. We suggest that the spacing of nucleosomal arrays is determined by interaction between adjacent histone octamers under physiological assembly conditions.     

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.     

Efficient human sperm pronucleus formation and replication in Xenopus egg extracts.

We have achieved efficient in vitro reactivation and replication of human sperm nuclei in frog egg extracts by constructing a 4-step protocol that mimics the events of fertilization and pronucleus formation in mammalian eggs. With use of this protocol, 78-97% of human sperm nuclei from fertile donors synchronously swelled and completed full genome replication in about 2 h. We document the changes in nuclear structure that accompany efficient DNA synthesis and discuss future research and potential clinical implications of this new system.     

Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts

The composition and function of histone storage complexes of Xenopus eggs have been investigated using monoclonal antibodies. We show that core histones are contained in two distinct complexes: H2A and H2B are associated with nucleoplasmin, and H3 and H4 are associated with nuclear protein N1. Immunodepletion analyses demonstrate that both complexes are required for nucleosome core assembly by extracts in vitro, the product being a simple sum of the histones from each complex. In addition, the majority of the stored H2A is shown to be an unusual form that migrates close to the position of H3 by SDS-polyacrylamide gel electrophoresis and resembles a variant synthesized in a cell-cycle-independent manner in mammalian cells.     

Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.     

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.