Maturation Spindle Research Articles

Longitudinal Analysis of Sleep Spindle Maturation from Childhood through Late Adolescence.

Sleep spindles are intermittent bursts of 11-15 Hz EEG waves that occur during non-rapid eye movement sleep. Spindles are believed to help maintain sleep and to play a role in sleep-dependent memory consolidation. Here we applied an automated sleep spindle detection program to our large longitudinal sleep EEG dataset (98 human subjects, 6-18 years old, >2000 uninterrupted nights) to evaluate maturational trends in spindle wave frequency, density, amplitude, and duration. This large dataset enabled us to apply nonlinear as well as linear age models, thereby extending the findings of prior cross-sectional studies that used linear models. We found that spindle wave frequency increased with remarkable linearity across the age range. Central spindle density increased nonlinearly to a peak at age 15.1 years. Central spindle wave amplitude declined in a sigmoidal pattern with the age of fastest decline at 13.5 years. Spindle duration decreased linearly with age. Of the four measures, only spindle amplitude showed a sex difference in dynamics such that the age of most rapid decline in females preceded that in males by 1.4 years. This amplitude pattern, including the sex difference in timing, paralleled the maturational pattern for δ (1-4 Hz) wave power. We interpret these age-related changes in spindle characteristics as indicators of maturation of thalamocortical circuits and changes in sleep depth. These robust age-effects could facilitate the search for cognitive-behavioral correlates of spindle waveforms and might also help guide basic research on EEG mechanisms and postnatal brain maturation.SIGNIFICANCE STATEMENT The brain reorganization of adolescence produces massive changes in sleep EEG. These changes include the morphology and abundance of sleep spindles, an EEG marker of non-rapid eye movement sleep believed to reflect offline memory processes and/or protection of the sleep state. We analyzed >2000 nights of longitudinal sleep EEG from 98 subjects (age 6-18 years old) to investigate maturational changes in spindle amplitude, frequency, density, and duration. The large dataset enabled us to detect nonlinear as well as linear age changes. All measures showed robust age effects that we hypothesize reflect the maturation of thalamocortical circuits and decreasing sleep depth. These findings could guide further research into the cognitive-behavioral correlates of sleep spindles and their underlying brain mechanisms.

SPINDLE CELL LIPOMA OF THE ORAL CAVITY: CASE REPORT OF AN UNCOMMON AND DISTINCT VARIANT OF LIPOMA

Spindle cell lipoma (SCL), an uncommon histologic variant of lipoma, is a benign tumor that often occurs in the posterior neck or shoulder with rare occurrence in the oral cavity. It comprises about 1.6% of intraoral lipomatous tumors. The aim of the case report is to present the histopathologic features of a SCL. A 60-year-old woman with noncontributory medical history was examined for an asymptomatic nodule in the left buccal mucosa. The intraoral examination showed a 7-mm sessile and well-circumscribed nodule with firm consistency and smooth surface covered by slightly whitish mucosa. Excisional biopsy was carried out based on a clinical diagnosis of inflammatory fibrous hyperplasia. The histopathology demonstrated mature adipose tissue, spindle cells (CD34+), and many mast cells in a connective tissue stroma. The diagnosis of SCL was hence established. The patient is being followed up.

Week-by-week changes in sleep EEG in healthy full-term newborns.

To extract independent EEG broad bands using principal component analysis (PCA) and describe week-by-week EEG changes in quiet sleep (QS) and active sleep (AS) during the first 5 weeks of postnatal life in healthy, full-term newborns. Polysomnography of spontaneous sleep was recorded in 60 newborns in 5 groups at 41, 42, 43, 44, and 45 weeks (n = 12 each) postconceptional age (POST-C). QS and AS stages were identified. Absolute power (AP) for 1 Hz bins between 1 and 30 Hz was subjected to PCA to extract independent broad bands. PCA rendered three independent broad bands distinct from conventional bands. They explained 82.8% of variance: 2-10 Hz, 10-16 Hz, and 17-30 Hz. ANOVAs (group × age × derivations) showed significant higher power at 2-10 Hz with greater age, higher power in QS than AS in all three bands, and significantly higher AP in the left central region, and in the right occipital and temporal areas, in both sleep stages. A different method of analyzing sleep EEG generated new information on brain maturation. The Sigma frequencies identified suggest that sleep spindle maturation begins by at least 41 weeks of POST-C age. Interhemispheric asymmetries during sleep suggest earlier development of the central left region and the right occipital and temporal areas.

HSP70 is required for the proper assembly of pericentriolar material and function of mitotic centrosomes

BackgroundAt the onset of mitosis, the centrosome expands and matures, acquiring enhanced activities for microtubule nucleation and assembly of a functional bipolar mitotic spindle. However, the mechanisms that regulate centrosome expansion and maturation are largely unknown. Previously, we demonstrated in an immortalized human cell line CGL2 and cancer cell line HeLa that the inducible form of heat shock protein 70 (HSP70) accumulates at the mitotic centrosome and is required for centrosome maturation and bipolar spindle assembly.ResultsIn this study, we further show that HSP70 accumulated at the spindle pole in a PLK1-dependent manner. HSP70 colocalized with pericentrin (PCNT), CEP215 and γ-tubulin at the spindle pole and was required for the 3D assembly of these three proteins, which supports mitotic centrosome function. Loss of HSP70 disrupted mitotic centrosome structure, reduced pericentriolar material recruitment and induced fragmentation of spindle poles. In addition, HSP70 was necessary for the interaction between PCNT and CEP215 and also facilitated PLK1 accumulation and function at the spindle pole. Furthermore, we found that HSP70 chaperone activity is required for PCNT accumulation at the mitotic centrosome and assembly of mitotic spindles.ConclusionOur current results demonstrate that HSP70 is required for the accurate assembly of the pericentriolar material and proper functioning of mitotic centrosomes.

Developmental changes of sleep spindles and their impact on sleep-dependent memory consolidation and general cognitive abilities: A longitudinal approach.

Sleep spindles are related to sleep‐dependent memory consolidation and general cognitive abilities. However, they undergo drastic maturational changes during adolescence. Here we used a longitudinal approach (across 7 years) to explore whether developmental changes in sleep spindle density can explain individual differences in sleep‐dependent memory consolidation and general cognitive abilities. Ambulatory polysomnography was recorded during four nights in 34 healthy subjects (24 female) with two nights (baseline and experimental) at initial recording (age range 8–11 years) and two nights at follow‐up recording (age range 14–18 years). For declarative learning, participants encoded word pairs with a subsequent recall before and after sleep. General cognitive abilities were measured by the Wechsler Intelligence Scale. Higher slow (11–13 Hz) than fast (13–15 Hz) spindle density at frontal, central, and parietal sites during initial recordings, followed by a shift to higher fast than slow spindle density at central and parietal sites during follow‐up recordings, suggest that mature spindle topography develops throughout adolescence. Fast spindle density increases from baseline to experimental night were positively related to sleep‐dependent memory consolidation. In addition, we found that the development of fast spindles predicted the improvement in memory consolidation across the two longitudinal measurements, a finding that underlines a crucial role for mature fast spindles for sleep‐dependent memory consolidation. Furthermore, slow spindle changes across adolescence were related to general cognitive abilities, a relationship that could indicate the maturation of frontal networks relevant for efficient cognitive processing. A video abstract of this article can be viewed at: https://www.youtube.com/watch?v=7NXJzm8HbIw and https://www.youtube.com/watch?v=iuMQY1OIJ0s

Different maturational changes of fast and slow sleep spindles in the first four years of life

Objective/BackgroundMassive changes in brain morphology and function in the first years of life reveal a postero-anterior trajectory of cortical maturation accompanied by regional modifications of NREM sleep. One of the most sensible marker of this maturation process is represented by electroencephalographic (EEG) activity within the frequency range of sleep spindles. However, direct evidence that these changes actually reflect maturational modifications of fast and slow spindles still lacks.Our study aimed at answering the following questions:1. Do cortical changes at 11.50 Hz frequency correspond to slow spindles?2. Do fast and slow spindles show different age trajectories and different topographical distributions?3. Do changes in peak frequency explain age changes of slow and fast spindles? Patients/MethodsWe measured the antero-posterior changes of slow and fast spindles in the first 60 min of nightly sleep of 39 infants and children (0–48 mo.). ResultsWe found that (A) changes of slow spindles from birth to childhood mostly affect frontal areas (B) variations of fast and slow spindles across age groups go in opposite direction, the latter progressively increasing across ages; (C) this process is not merely reducible to changes of spindle frequency. ConclusionsAs a main finding, our cross-sectional study shows that the first form of mature spindle (i.e., corresponding to the adult phasic event of NREM sleep) is marked by the emergence of slow spindles on anterior regions around the age of 12 months.

Posterior Neck Triangle Spindle Cell Lipoma

Spindle cell lipomas (SCLs) are a rare variant of lipomas with distinct pathological and immunohistological features. Pathological characteristics of SCLs have been well described and include the following features: a well-circumscribed subcutaneous lesion that contains a variety of mature adipocytes, spindle cells and collagen bundles. Immunohistochemical staining of SCLs is usually positive for CD34. Four cases of posterior neck triangle SCLs have been reported in the literature. We present a further case of SCL presenting in the posterior triangle of the neck. A 51-year-old man with a large right posterior neck triangle mass was referred to our Head & Neck Clinic. Further questioning revealed that this swelling had been growing over a two and a half year period associated with neck pain, difficulty swallowing and recurrent skin infections at the base of the mass. Due to the rare nature of SCLs, such cases should be discussed in a dedicated multi-disciplinary team meeting. Although metastasis from SCLs is rare, experienced pathologists and radiologists should be consulted early in order to distinguish the type of lesion and the risk of metastasis from more sinister lesions such as liposarcoma. Surgical excision of SCLs is currently accepted as the gold standard and mainstay of management. J Med Cases. 2016;7(6):208-212 doi: http://dx.doi.org/10.14740/jmc2462w

Centrosomin represses dendrite branching by orienting microtubule nucleation.

Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization.

Shedding Light on Mysterious Microtubules

We have known for decades that vertebrate kinetochores can nucleate microtubules. However, the role of such microtubules was unclear. Kitamura et al. investigated this issue by following a marked budding yeast centromere after its conditional reactivation to examine microtubule capture at unattached kinetochores. They found not only that yeast kinetochores can nucleate microtubules, but also that such microtubules facilitate attachment by decreasing the time required for spindle-pole-associated microtubules to make contact with unattached kinetochores. Historically, the fact that microtubules nucleated at kinetochores are opposite in polarity to those in mature spindle fibers was used to argue that they were not physiologically relevant for spindle assembly. Kitamura et al. have now shown that they, in fact, do play a role, and then rapidly depolymerize after spindle fiber attachments form. In sum, this paper provides a function for a previously mysterious microtubule population and outlines a surprising and dynamic mechanism through which kinetochore-originated microtubules assist spindle pole microtubules to efficiently locate unattached kinetochores. It also shows how this assistance mechanism is shut off to dim the locator beacon after correct attachments have been made. Understanding Kinetochore-Nucleated Microtubules Tomoyuki Tanaka, with the help of Etsushi Kitamura and Shinya Komoto, explains his group's key findings regarding the roles of kinetochore-generated microtubules in mitosis.

New Strategies for Anterior Cruciate Ligament Partial Rupture Using Bone Marrow Transplantation in Rats

The purpose of this study was to compare anterior cruciate ligament (ACL) regeneration between animal groups subjected to intra-articular injection of fresh whole bone marrow cells (BMCs), cultured mesenchymal stem cells (MSCs), or saline. Partially transected ACLs in Fischer 344/Nslc rats were prepared, followed by injection of BMCs, MSCs, or saline into the articular cavity at 1 week after transection. Donor cells expressing green fluorescent protein were detected in the recipient's transected ACLs at 4 weeks in the BMC and MSC groups, and their ACLs appeared almost normal histologically. Further, there were significantly more mature spindle cells in the BMC group than in the saline group at 4 weeks. Biomechanically, the tensile strength in the BMC group reached near normal levels at 4 weeks after injection. The levels of transforming growth factor-β1 in the ACL tissue and knee joint fluid in the BMC group were increased significantly compared with that of the saline group at 4 weeks as detected by immunohistochemical analysis. In conclusion, intra-articular bone marrow transplantation using fresh whole BMCs is an effective treatment for ACL partial rupture. This therapy is easy to apply in a clinical setting because no culture system is required for collecting MSCs.

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle.

1-Naphthaleneacetic acid and 6-benzyladenine thinning of a common slender spindle ‘Jonagold’/M.9 apple orchard. I: Dose effects and spray distribution in the crowns

SummaryTo find the most appropriate rates of application of plant growth regulator (PGR) thinning-agents for a common slender spindle apple (Malus × domestica Borkh.) orchard, different volumes of dilute 1-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) were sprayed ha−1. Mature ‘Jonagold’/M.9 trees, 3.0 – 3.5 m high and 1.2 – 1.5 m wide, planted in a single row system with 3,030 trees ha−1, were used. Significant thinning was observed in the case of dilute sprays of NAA at 10 mg l−1, or BA at 100 mg l−1, to run-off, using 2,000 l ha−1 or 1,500 l ha−1; while 1,000 l ha−1 did not result in sufficient thinning. Thinning using smaller volumes (250, 500, or 750 l ha−1) was also significant if the concentration of PGR thinner was proportionally higher (i.e., based on the 1,500 l ha−1 application rate of more dilute sprays of NAA at 10 mg l−1 or BA at 100 mg l−1). Spray distribution measurements in the crowns showed better spray deposits when higher water volumes (i.e., more dilute PGR solutions) were sprayed at all positions (bottom, middle, or top) of the canopy. At 2,000 l ha−1, 54 – 72% coverage of the leaf area was observed; but, at 250 l ha−1, coverage was only 10 – 21%. The lower 30% of the canopy was covered poorly when smaller volumes of water (250, 500, or 1,000 l ha−1) were applied. When 1,500 l ha−1 was sprayed, good coverage of the lower and upper surfaces of the leaves occurred, and no differences in canopy positions were measured. It was concluded that 1,500 l ha−1 (i.e., dilute PGR) spraying was the most appropriate volume to use when calculating the dose of NAA or BA to be applied ha−1 to common (3.0 – 3.5 m-high) mature slender spindle apple orchards on M.9 rootstock. This study was part of the ISAFRUIT Smartfruit Project, aimed at improving existing methods for apple crop regulation with more precise use of PGR thinning agents and with minimum impact on the environment.

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore-microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.

The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis.

Degradation of the mitotic cyclins is a hallmark of the exit from mitosis. Induction of stable versions of each of the three mitotic cyclins of Drosophila, cyclins A, B, and B3, arrests mitosis with different phenotypes. We tested a recent proposal that the destruction of the different cyclins guides progress through mitosis. Real-time imaging revealed that arrest phenotypes differ because each stable cyclin affects specific mitotic events differently. Stable cyclin A prolonged or blocked chromosome disjunction, leading to metaphase arrest. Stable cyclin B allowed the transition to anaphase, but anaphase A chromosome movements were slowed, anaphase B spindle elongation did not occur, and the monooriented disjoined chromosomes began to oscillate between the spindle poles. Stable cyclin B3 prevented normal spindle maturation and blocked major mitotic exit events such as chromosome decondensation but nonetheless allowed chromosome disjunction, anaphase B, and formation of a cytokinetic furrow, which split the spindle. We conclude that degradation of distinct mitotic cyclins is required to transit specific steps of mitosis: cyclin A degradation facilitates chromosome disjunction, cyclin B destruction is required for anaphase B and cytokinesis and for directional stability of univalent chromosome movements, and cyclin B3 degradation is required for proper spindle reorganization and restoration of the interphase nucleus. We suggest that the schedule of degradation of cyclin A, cyclin B, and then cyclin B3 contributes to the temporal coordination of mitotic events.

Dominant human herpesvirus type 8 RNA transcripts in classical and AIDS-related Kaposi's sarcoma.

Skin biopsy sections of Kaposi's sarcoma (KS) from 25 patients (5 AIDS-related, 20 classical cases) were histologically staged and hybridized in situ with oligonucleotide probes for constitutively transcribed human herpesvirus 8 (HHV-8) mRNA T0.7 and T1.1 using a colourimetric technique. T1.1 increases during experimental induction of the viral lytic phase in the HHV-8-infected lymphocytes of primary effusion lymphoma and its colourimetric detection in KS cells presumably corresponds to virion production. Immunostaining with anti-CD20, CD45RO, MAC 387, and alpha-smooth muscle actin was performed following T1.1 in situ hybridization (ISH). When the amount of T0.7 was above the detection threshold, the signal was made up of multiple coarse intranuclear dots in most spindle cells. Of the six early-stage lesions, none produced a T1.1 hybridization signal. Two of four AIDS-related and two of eight classical lesions with incipient spindle cell growth produced rare but distinct dense intranuclear T1.1 signals in endothelial cells lining narrow tubes. In contrast, eight of ten (all classical KS) mature spindle cell lesions displayed a signal, scattered in up to 2 per cent of spindled endothelial cells. Cell types other than endothelium produced no T1.1 hybridization signal in double stains. The results are consistent with other published data indicating latent HHV-8 infection in endothelium and its tumour cell progeny, with simultaneous virion production in a small subset of cells. Immunodeficiency may not influence the number of cells lytically infected with HHV-8 in early KS, in contradistinction to other herpesviruses with latent-lytic cycles.

Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries.

The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.

Measurement using a non-contact 3-D measurement system and histopathologic study of surface of skin scar formation after cheiloplasty

As to postoperative scar formation of maxillofacial lesions, a number of authors have reported on the clinical classification and light or electron microscopic observations of hypertrophic scars. However, there has been no quantitative study to evaluate the surface of the postoperative scar and its relevance to changes in histopathology. The purpose of this study was to assess postoperative scar formation on patients with cleft lip quantitatively by using a non-contact 3-D measurement system and to compare the findings with the histopathologic changes. Thirty-six specimens were obtained from patients when they underwent corrective surgery of the lip. The depth of the scar was measured by using a non-contact 3-D measurement system. The results were compared with morphological findings of fibroblasts and collagen fibers of the scar tissues under a light microscope. The results were following as follows: 1. The depth of the CLP group was 181.4 +/- 78.4 microns, significantly higher than 55.7 +/- 24.8 microns of the non-cleft group (p < 0.05). 2. Histologically, the scar, which formed a diffuse mass of irregularly arranged thick collagen fibers, showed a higher value in the depth of the scar than the one which formed a regular pattern. 3. The scar with mature spindle fibroblasts showed a higher value in the depth of the scar than the one with immature round fibroblasts. In conclusion, it was suggested that a non-contact 3-D measurement system is useful for measuring the depth of the scar surface, and the histologic changes of the scar influence the depth.

Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage.

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 microm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a "start anaphase" reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Electron microscope observations on LH-induced oocyte maturation in Japanese quail (Coturnix coturnix japonica)

The aim of this study was to describe the temporal sequence of ultrastructural changes in the boundary between the preovulatory oocyte and its surrounding follicular wall during maturation induced by injection of LH. Female Japanese quail were injected with ovine LH (20 micrograms per bird) 10-12 h before the expected time of ovulation. The largest and second largest follicles were excised before or 1, 2, 4 or 6 h after injection. The oocyte and the surrounding follicular wall were processed for observations using light and electron microscopy. Before injection of LH, cytoplasmic projections of granulosa cells interdigitated with microvilli on the surface of the oocyte and formed spot desmosomes and gap junctions with the oolemma. Two hours after injection of LH, the germinal vesicles in the largest but not in the second largest follicles began to break down and membrane-bound vesicles increased in number and size in the surrounding germinal disc. The junctions between the oocyte surface and the granulosa cell projections started to dissociate and a perivitelline space began to develop, possibly as the result of an accumulation of fluids transported from the capillary sinus in the theca interna. The first maturation spindle was formed 4 h after injection of LH, whereas the first polar body and the second maturation spindle were formed 6 h after LH stimulation. These observations suggest that the dissociation of connections between the oocyte and granulosa cells 2 h after exposure to increased concentration of LH is the first process of oocyte maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Okadaic Acid Induces Spindle Lengthening and Disrupts the Interaction of Microtubules with the Kinetochores in Metaphase II-Arrested Mouse Oocytes

The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 μ M OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immuno-blotting and [ 32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.