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PubMed2005

Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy.

BACKGROUND: Despite recent technical improvements, many human preimplantation embryos fail to develop to the blastocyst stage or implant after transfer to the uterus. A possible cause for this developmental arrest is the high incidence of nuclear and postzygotic chromosomal abnormalities observed during cleavage, including chaotic chromosome complements, suggestive of defects in mitotic chromosomal segregation. The underlying mechanisms are largely unknown, but similarities with chromosome instability in human cancers led to the proposal that cell cycle checkpoints may not operate at these early stages. METHODS: To investigate this and to examine whether spindle abnormalities contribute to chromosome malsegregation, we have used fluorescence and confocal laser scanning microscopy, following immunolabelling with antibodies specific for alpha-tubulin, gamma-tubulin, or acetylated tubulin, combined with a DNA fluorochrome to visualize nuclei, spindle and chromosome configurations in normal and arrested human embryos, from cleavage to blastocyst stages. RESULTS: In addition to frequent interphase nuclear abnormalities, we identify for the first time various spindle abnormalities including abnormal shape and chromosome loss and multipolar spindles at cleavage and blastocyst stages. CONCLUSIONS: We propose that a major pathway leading to postzygotic chromosomal abnormalities is the formation of binucleate blastomeres with two centrosomes which result either in a bipolar spindle and division to two tetraploid blastomeres, or in a multipolar spindle, chromosome malsegregation and chromosomal chaos.

Acetylation↗

PubMed2005

Bisphenol-A induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis.

Bisphenol-A (BPA) is a widely used environmental estrogen-like chemical that has a weak estrogenic activity. This study aimed to test the potential inhibitory effects of BPA on meiotic cell cycle progression, centrosomes and spindle integrity in mouse cumulus-oocyte complexes (COCs). They were exposed to BPA (10-30 microM; 2.3-6.8 ppm) during meiosis-I and the formation of metaphase-II (M-II) spindle. Exposure to BPA during meiosis-I caused a dose-dependent retardation/inhibition of cell cycle progression; 74 and 61% of cells reached metaphase-I (M-I) in the presence of 10 and 30 microM BPA, respectively, (81% in controls, P<0.001). A more striking delay was noted when oocytes were exposed to BPA during the formation of M-II spindle, i.e. 61 and 41% of cells (94% in controls, P<0.001) reached M-II while the remaining cells remained at M-I. Depending on dose, both (i) loosening and elongation of meiotic spindles and (ii) compaction and dispersion of pericentriolar material (PCM) were noted in all samples, all of which resulted in a series of spindle abnormalities. Interestingly, no chromosome was detected in the first polar body after the 10 and 30 microM BPA treatments. When the cells were freed from BPA exposure at 10 and 30 microM, 70 and 61%, of the cells succeeded in reaching M-II (93% in controls, P<0.001), respectively. In conclusion, one mode of action of BPA is a moderately severe yet reversible delay in the meiotic cell cycle, possibly by a mechanism that degrades centrosomal proteins and thus perturbs the spindle microtubule organization and chromosome segregation.

Animals↗

PubMed1992

In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle.

Cryopreservation of germinal vesicle (GV)-stage mouse oocytes results in a developmental block. As an approach to explain the failure in development, we have investigated the morphology of the second meiotic spindle after in-vitro maturation. Fully grown GV-stage mouse oocytes were collected from the ovaries of primed mice and kept in meiotic arrest with dibutyryl cyclic AMP. These oocytes were submitted to different variables of cryopreservation: (i) cooling to 22 degrees C or 0 degrees C; (ii) exposure to 1.5 M 1,2-propanediol at 22 degrees C or 0 degrees C; (iii) exposure to 1.5 M dimethylsulphoxide (DMSO) at 22 degrees C or 0 degrees C; (iv) ultrarapid freezing with 3.5 M DMSO/0.5 M sucrose; (v) exposure to a sucrose dehydration series according to the ultrarapid freezing protocol. The morphology of the second meiotic spindle was evaluated 16 h after release from meiotic arrest. We were able to demonstrate that following cooling, exposure to cryoprotectants or ultrarapid freezing of GV-stage mouse oocytes, a normal barrel-shaped spindle with the chromosomes in midplane position is found in 79-94% of oocytes except for two conditions with great exposure to dehydration stress. Exposure to DMSO at 0 degrees C or exposure to a sucrose dehydration series resulted in significantly lower percentages of barrel-shaped spindles, respectively 64% and 62%. The effect on spindle morphology has to be put into perspective, however, since the observed abnormalities were changes of spindle shape, such as elongation or reduction, which are assumed to be restorable, and since no polar organizational defects were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Decision of spindle poles and division plane by double preprophase bands in a BY-2 cell line expressing GFP-tubulin.

The preprophase band (PPB) of microtubules is thought to be involved in deciding the future division site. In this study, we investigated the effects of double PPBs on spindle formation and the directional decision of cytokinesis by using transgenic BY-2 cells expressing green fluorescent protein (GFP)-tubulin. At prophase, most of the cells with double PPBs formed multipolar spindles, whereas all cells with single PPBs formed normal bipolar spindles, clearly implicating the PPB in deciding the spindle poles. At metaphase, however, both cell types possessed the bipolar spindles, indicating the existence of correctional mechanism(s) at prometaphase. From prometaphase to metaphase, the spindles in double PPB cells altered their directions to become oblique to the cell-elongating axis, and these orientations were maintained in the phragmoplast and resulted in the oblique division planes. These oblique cell plates decreased when actin microfilaments were disrupted, and double actin-depleted zones (ADZs) appeared where the double PPBs had existed. These results suggest that the information necessary for proper cytokinesis may be transferred from the PPBs to the ADZs, even in the case of the double PPBs.

Actin Cytoskeleton↗

PubMed2007

2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes.

2-Methoxyestradiol (2-ME) is a metabolite of 17beta-estradiol and a natural component of follicular fluid. Local concentrations of 2-ME may be increased by exposure to environmental pollutants that activate the expression of enzymes in the metabolic pathway from 17beta-estradiol to 2-ME. It has been suspected that this may have adverse effects on spindle formation in maturing oocytes, which would affect embryo quality. To study the dose-response patterns, we exposed denuded mouse oocytes to 2-ME during in vitro maturation. Meiotic progression, spindle morphology, centrosome integrity, and chromosome congression were examined by immunofluorescence and noninvasive polarizing microscopy (PolScope). Chromosomal constituents were assessed after spreading and C-banding. 2-ME sustained MAD2L1 expression at the centromeres and increased the number of meiosis I-blocked oocytes in a dose-dependent manner. 2-ME also caused dramatic dose-dependent increases in the hyperploidy of metaphase II oocytes. Some of these meiosis II oocytes contained anaphase I-like chromosomes, which suggests that high concentrations of the catecholestradiol interfere with the physical separation of chromosomes. Noninvasive PolScope analysis and tubulin immunofluorescence revealed that perturbations in spindle organization, which resulted in severe disturbances of the chromosome alignment at the spindle equator (congression failure), were caused by 2-ME at meiosis I and II. Pericentrin-positive centrosomes failed to align at the spindle poles, and multipolar spindles and prominent arrays of cytoplasmic microtubule asters were induced in 2-ME-exposed metaphase II oocytes. In conclusion, a micromolar level of 2-ME is aneugenic for mammalian oocytes. Therefore, exposure to 2-ME and conditions that increase the intrinsic local concentration of 2-ME in the ovary may affect fertility and increase risks for chromosomal aberrations in the oocyte and embryo.

2-Methoxyestradiol↗

PubMed1994

Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes.

The purpose of this study was to evaluate effects of cooling and rewarming on the meiotic spindle apparatus of bovine oocytes. In experiment 1, in vitro-matured bovine oocytes were either maintained at 39 degrees C or cooled abruptly to 4 degrees C or approximately 25 degrees C. Immunohistochemical and DNA staining for visualization of microtubules and chromosomes, respectively, revealed an anastral, barrel-shaped spindle in bovine oocytes. Exposure to 4 degrees C for 10-20 min caused complete disappearance of the spindle. Some chromosome dispersion occurred after 60 min at 4 degrees C. After exposure to approximately 25 degrees C for 30 min, 90% of oocytes appeared abnormal, having either an abnormal spindle or no spindle. In experiment 2, oocytes cooled to either approximately 25 degrees C or 4 degrees C for 30 min were rewarmed directly or in steps for 15 or 60 min. Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme. Step-wise rewarming was no more beneficial than direct rewarming. More of the oocytes rewarmed directly contained dispersed chromosomes as time at 39 degrees C increased.

Animals↗

PubMed2003

A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C elegans.

The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Here, we describe two closely related proteins, GPR-1 and GPR-2 (G protein regulator), which associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Galpha(i/o). Inactivation of lin-5, gpr-1/gpr-2, or the Galpha(i/o) genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Galpha locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Galpha(i/o) signaling.

Animals↗

PubMed1998

Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis.

We analyzed male meiosis in mutants of the chickadee (chic) locus, a Drosophila melanogaster gene that encodes profilin, a low molecular weight actin-binding protein that modulates F-actin polymerization. These mutants are severely defective in meiotic cytokinesis. During ana-telophase of both meiotic divisions, they exhibit a central spindle less dense than wild type; certain chic allelic combinations cause almost complete disappearance of the central spindle. Moreover, chic mutant spermatocytes fail to form an actomyosin contractile ring. To further investigate the relationships between the central spindle and the contractile ring, we examined meiosis in the cytokinesis-defective mutants KLP3A and diaphanous and in testes treated with cytochalasin B. In all cases, we found that the central spindle and the contractile ring in meiotic ana-telophases were simultaneously absent. Together, these results suggest a cooperative interaction between elements of the actin-based contractile ring and the central spindle microtubules: When one of these structures is disrupted, the proper assembly of the other is also affected. In addition to effects on the central spindle and the cytokinetic apparatus, we observed another consequence of chic mutations: A large fraction of chic spermatocytes exhibit abnormal positioning and delayed migration of asters to the cell poles. A similar phenotype was seen in testes treated with cytochalasin B and has been noted previously in mutants at the twinstar locus, a gene that encodes a Drosophila member of the cofilin/ADF family of actin-severing proteins. These observations all indicate that proper actin assembly is necessary for centrosome separation and migration.

Actins↗

PubMed1995

p93dis1, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites.

Fission yeast cold-sensitive (cs) dis1 mutants are defective in sister chromatid separation. The dis1+ gene was isolated by chromosome walking. The null mutant showed the same phenotype as that of cs mutants. The dis1+ gene product was identified as a novel 93-kD protein, and its localization was determined by use of anti-dis1 antibodies and green fluorescent protein (GFP) tagged to the carboxyl end of p93dis1. The tagged p93dis1 in living cells localizes along cytoplasmic microtubule arrays in interphase and the elongating anaphase spindle in mitosis, but association with the short metaphase spindle microtubules is strikingly reduced. In the spindle, the tagged p93dis1 is enriched at the spindle pole bodies (SPBs). Time-lapse video images of single cells support the localization shift of p93dis1 to the SPBs in metaphase and spindle microtubules in anaphase. The carboxy-terminal fragment, which is essential for Dis1 function, accumulates around the mitotic SPB. We propose that these localization shifts of p93dis1 in mitosis facilitates sister chromatid separation by affecting SPB and anaphase spindle function.

Amino Acid Sequence↗

PubMed2007

The kinesin ATK5 functions in early spindle assembly in Arabidopsis.

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.

Arabidopsis↗

PubMed2005

Modulated targeting of GFP-AtMAP65-1 to central spindle microtubules during division.

AtMAP65-1 bundles cortical microtubules and we examined how this property is regulated during division in time-lapse studies of Arabidopsis suspension cells expressing GFP-AtMAP65-1. Spindle fluorescence is diffuse during metaphase, restored to the central spindle at anaphase and then compacted at the midline during late anaphase/early telophase. However, mutagenesis of the microtubule-associated protein (MAP) consensus Cdk site to a non-phosphorylatable form allows premature decoration of microtubules traversing the central region of the metaphase spindle without affecting the timing of the subsequent compaction. This suggests that mutagenesis does not affect compaction but does affect a phosphorylation/dephosphorylation switch that normally targets AtMAP65-1 to the central spindle at the metaphase/anaphase transition. GFP-AtMAP65-1 continues to label the midline of the early phragmoplast, suggesting a structural continuity with the central spindle - both structures being composed of anti-parallel microtubules. However, once the cytokinetic apparatus expands into a ring the MAP becomes depleted at the midline. Despite this, cytokinesis is not arrested and membrane and callose are deposited at the cell plate. It is concluded that AtMAP65-1 plays a role in the central spindle at anaphase to early cytokinesis but is not essential at the midline of the phragmoplast at later stages.

Arabidopsis↗

PubMed1975

The incidence and properties of beta axons to muscle spindles in the cat hind limb.

The distribution of beta axons to muscle spindles in the tenuissimus and abductor digiti quinti medius (A.D.Q.M.) muscles of the hind limb of the cat was determined by testing the action of single motor axons, capable of producing extrafusal contraction, isolated in the ventral spinal roots on the discharges of individual muscle spindle primary sensory endings recorded in the dorsal spinal roots. The proportion of spindles with beta innervation was 41% in A.D.Q.M. and 30% in tenuissimus. The proportion of fast motor axons that were beta axons was 28% in the A.D.Q.M. and 11% in tenuissimus; usually each beta axon innervated a single spindle while no spindle received more than two beta axons. The beta axons were dynamic in nature and those to any one muscle tended to have slightly lower conduction velocities than the alpha axons though some overlap did occur. The extent to which beta axons can account for the fact that in isolated spindles axons selective to either nuclear bag or nuclear chain fibres are found in about equal proportions whereas a ratio of three static to one dynamic gamma axons is found electrophysiologically is discussed. An explanation for the low incidence of beta innervation previously found electrophysiologically and the considerably higher incidence found histologically is given.

Action Potentials↗

PubMed1968

The response to stretch of muscle spindle afferents of baboon's tibialis anticus and the effect of fusimotor stimulation.

1. Systematic studies of the response of baboon's tibialis anticus muscle spindles to stretch were undertaken. Most of the spindle afferents studied had conduction velocities between 72 and 78 m/sec with a range from 39 to 93 m/sec. There was no clear bimodality in the histogram of the conduction velocity. Measurements were made of the axon diameters of the nerve to tibialis anticus. The largest number of the larger axons had diameters from 11 to 13 mu. The largest axon diameter measured 17 mu and there were very few of these.2. The dynamic index for any given afferent tended to be greater for the more rapidly conducting afferents and lower for the more slowly conducting afferents. However, a statistically significant regression line of this relationship could only be drawn for a stretch velocity of 64 mm/sec.3. The spindle afferent response to stretch was studied from different muscle lengths. It was found that the velocity sensitive portion of the response to phasic stretch decreased when the stretch extended up to or beyond the maximum physiological length of the muscle.4. Dynamic and static fusimotor fibres were isolated. The response of the spindle afferent to stretch, while the dynamic fusimotor fibre was being stimulated, was the same as that reported for the cat by P. B. C. Matthews (1962). During static fusimotor stimulation the response of the spindle afferent to stretch was usually like that reported for the cat. In a single case, however, static fusimotor stimulation resulted in a lowering of the peak frequency of the response of the spindle afferent to the phasic portion of stretch.

Action Potentials↗

PubMed1969

An electrophysiological analysis of responses from lizard muscle spindles.

1. Stretch receptor discharges were recorded in the lizard Tiliqua nigrolutea. Responses were identified as coming from muscle spindles by their ;in parallel' behaviour during a twitch.2. Recordings from spindles in the iliofibularis muscle which contains many single innervated fibres revealed spindles which discharged on the rising phase of the muscle contraction. This ;in series' response increased in frequency and duration at high resting tensions on the muscle and was attributed to a specific intrafusal contraction.3. The ;in series' discharge could be induced by stimulation of only one small filament of the muscle nerve. This suggested a focal intrafusal motor innervation.4. Responses from spindles in the semitendinosus muscle, which contains many multiple innervated fibres provided examples of an ;in series' response which could be produced by stimulation of several muscle nerve filaments. This was interpreted as being an example of multiple intrafusal motor innervation.5. Spindles with short or elongated sensory regions were subjected to ;ramp' stretches. The presence of distinct phasic and tonic responses from the two types was confirmed and related to the behaviour of mammalian spindles.

Animals↗

PubMed1975

An investigation into the site of termination of static gamma fibres within muscle spindles of the cat peroneus longus muscle.

1. The distribution of static fusimotor fibres to intrafusal muscle fibres of cat peroneus longus muscle spindles was investigated using the glycogen-depletion technique of Edström & Kugelberg (1968). Single static gamma fibres were stimulation intermittently at high rates for 3 hr with the blood supply occluded for some of this time. Subsequently the portion of muscle containing the activated spindles was fixed, sectioned and stained for glycogen with the periodic acid-Schiff (PAS) method. 2. Ten static axons caused depletion in eleven spindles. In five of these the only glycogen-depleted fibres were nuclear chain fibres. In the other six spindles one nuclear bag fibre was depleted in addition to chain fibres and this was always the larger of the two within the spindle. 3. These results on a medium-sized hind limb muscle are compared with findings concerning the distribution of static gamma fibre axons previously investigated only in very small muscles. The results agree in showing that nearly all static gamma fibres innervate nuclear chain fibres but that in 50-75% of the times in which static gamma fibres innervate spindles the distribution is to bag fibres as well as to chain fibres. The interpretation to be put upon this is uncertain. One possibility with which the results from peroneus longus are consistent is that the bag fibres which are usually innervated by static axons are the 'intermediate' bag fibres whose ultrastructure has recently been shown to resemble that of chain fibres.

Animals↗

PubMed1976

Distribution of fusimotor axons to intrafusal muscle fibres in cat tenuissimus spindles as determined by the glycogen-depletion method.

1. The distribution of fusimotor axons to bag1, bag2 and chain muscle fibres in cat tenuissimus spindles has been studied using a modification of the glycogen-depletion technique of Edstrrom & Kugelberg (1968). Single fusimotor axons were stimulated intermittently at 40-100/sec for long periods (30-90 sec) during blood occlusion. Portions of muscle containing the activated spindles were quick-frozen, fixed in absolute ethanol during freeze-substitution, and then embedded in paraffin wax. Serial transverse sections were stained for glycogen using the periodic acid-Schiff method, and examined for depletion. 2. Dynamic gamma axons (i.e. those that increase the dynamic index of primary-ending responses to ramp stretches of large amplitude) depleted bag1 fibres almost exclusively. 3. Static gamma axons (i.e. those that reduce or abolish the dynamic index) depleted both bag and chain fibres. Bag1 and bag2 fibres were depleted about equally. 4. A single static gamma axon may activate both bag and chain fibres in one spindle (the most common pattern), chain fibres only in another, and bag fibres only in a third spindle. 5. Static gamma axons with conduction velocities less than 25 m/sec also had a non-selective distribution, but no depletion was observed in bag2 fibres. 6. The zones of depletion produced by dynamic gamma axons were distributed more or less equally in the intra- and extracapsular parts of spindle poles, whereas those produced by static gamma axons were mainly intracapsular. 7. The results are compared with the glycogen-depletion studies of Brown & Butler (1973, 1975) and our own study of the distribution of static gamma axons to spindles in which all other motor axons had degenerated (Barker, Emonet-Dénand, Laporte, Proske & Stacey, 1973). The implications of the finding that both static gamma and dynamic gamma axons activate bag1 fibres are discussed.

Animals↗

PubMed1980

Induced changes in the thresholds for voluntary activation of human spindle endings.

1. Recordings were made from forty-two muscle spindle afferents from the pre-tibial muscles of human subjects. For each afferent, the contraction level at which its discharge accelerated (its 'threshold for activation') was defined using isometric voluntary contractions of the receptor-bearing muscle. The effects on these thresholds of various manoeuvres designed to activate descending pathways or segmental sensory inputs were studied to determine whether the balance between the skeletomotor and fusimotor drives to the contracting muscle could be altered. 2. The Jendrassik reinforcement manoeuvre raised the threshold for voluntary activation of one spindle ending slightly but had no significant effect with seven other endings. It is concluded that the Jendrassik manoeuvre does not excite fusimotor neurons selectively or even preferentially. 3. Caloric vestibular stimulation altered the thresholds for voluntary activation of eight of eleven spindle endings, the most common effect being lowering of the threshold during the irrigation and for 1--1 . 5 min after its cessation. 4. Cutaneous stimulation by vibration lowered the thresholds for voluntary activation of some spindle endings when applied to the dorsum of the foot, and raised thresholds when applied to the lateral plantar surface. 5. Passive stretch of the receptor-bearing muscle by plantar flexion of the ankle and passive stretch of synergistic muscles in isolation raised thresholds for spindle activation; passive stretch or vibration of triceps surae lowered thresholds. 6. These studies suggest that the relationship between the skeletomotor and fusimotor drives to a muscle during a voluntary contraction is not rigidly fixed, but can be varied appropriately with the changing motor role demanded of the muscle by supraspinal drives and with the changes in sensory feed-back generated by the movement itself. 7. It is concluded that, provided the skeletomotor effects of a stimulus are known, changes in the threshold for spindle activation in a voluntary contraction can provide a means of determining the effects of that stimulus on fusimotor neurons even when the stimulus alone is not adequate to alter fusimotor drive.

Ankle Joint↗

PubMed1986

Studies of capsule and capsular space of cat muscle spindles.

The presence of hyaluronate in the capsular space of the cat muscle spindle was demonstrated using alcian blue staining at various pHs, the critical electrolyte concentration technique and hyaluronidase treatment. In spindles with intact capsules an extracellular marker, the dye Ruthenium Red, gained access to the capsular space through the gap in the sleeve region, but for a limited distance. In muscle spindles with the capsule nicked, the marker diffused into the capsular space in the equatorial region, revealing a dense network in this space which consisted of globular structures interconnected by thin filaments. Based on their thickness, these filaments were inferred to be hyaluronic acid, and the globular structures were inferred to be protein molecules. Longitudinal diffusion of the dye into the capsular space through the nicked site was limited. The limited diffusion is probably due to electrostatic binding of the dye, which is a hexavalent cation, to negatively charged glycosaminoglycan hyaluronate that is present in the space. The transcapsular potential was measured by use of glass micropipettes filled with 3 M-KCl. The value was 15 mV +/- 4 (average +/- S.D., n = 12; range, 10-20 mV) inside negative. The input resistance and capacitance of the capsule, measured with two independent electrodes, varied widely (1.3-8.0 M omega and 0.5-1.3 nF, n = 4) and the capsule showed marked delayed rectification to outward current pulses. [K+] in the space measured with K+-sensitive resin-filled glass micropipettes was a few millimolar higher than that in the bathing solution. The effects of [K+] and [Ca2+] on impulse activities were examined in spindles with intact capsules or with partially resected capsules. In spindles with intact capsules the effects of [K+] and [Ca2+] were significantly less or negligible compared with those in spindles with the capsule opened. Hyaluronidase (approximately 10(-4) g/ml) added to the bathing solution around nicked capsules significantly reduced both resting and stretch-induced impulse activities in 40-50 min. By this time the capsular space was completely collapsed. An increase in [K+] of the bathing solution from 3.5 to 6 or 8 mM restored these impulse activities. A similar restoring effect was also observed when [Ca2+] in the bathing solution was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗