scholarly journals Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs.

1982 ◽  
Vol 94 (2) ◽  
pp. 455-465 ◽  
Author(s):  
G Schatten ◽  
H Schatten ◽  
T H Bestor ◽  
R Balczon
Keyword(s):  
Immunofluorescence Microscopy ◽  
De Novo ◽  
Sea Urchins ◽  
Microtubule Assembly ◽  
Lytechinus Variegatus ◽  
Sperm Tail ◽  
Arbacia Punctulata ◽  
Video Recordings ◽  
Transmission Electron ◽  
Sperm Aster

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.

scholarly journals MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

1965 ◽  
Vol 25 (2) ◽  
pp. 81-100 ◽  
Author(s):  
Luther E. Franklin
Keyword(s):  
Electron Microscope ◽  
Sea Urchin ◽  
Acrosome Reaction ◽  
Sea Water ◽  
Sea Urchins ◽  
Detailed Examination ◽  
Lytechinus Variegatus ◽  
Arbacia Punctulata ◽  
Sperm Entry ◽  
Ultrathin Sections

Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13).

scholarly journals Novel variants in DNAH9 lead to nonsyndromic severe asthenozoospermia

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Dongdong Tang ◽  
Yanwei Sha ◽  
Yang Gao ◽  
Jingjing Zhang ◽  
Huiru Cheng ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Sperm Morphology ◽  
Immunofluorescence Staining ◽  
Compound Heterozygous ◽  
Sperm Tail ◽  
Transmission Electron ◽  
Whole Exome ◽  
Detailed Medical History ◽  
Novel Variants ◽  
Common Causes

Abstract Background Asthenozoospermia is one of the most common causes of male infertility, and its genetic etiology is poorly understood. DNAH9 is a core component of outer dynein arms in cilia and flagellum. It was reported that variants of DNAH9 (OMIM: 603330) might cause primary ciliary dyskinesia (PCD). However, variants in DNAH9 lead to nonsyndromic severe asthenozoospermia have yet to be reported. Methods Whole exome sequencing (WES) was performed for two individuals with nonsyndromic severe asthenozoospermia from two non-consanguineous families, and Sanger sequencing was performed to verify the identified variants and parental origins. Sperm routine analysis, sperm vitality rate and sperm morphology analysis were performed according the WHO guidelines 2010 (5th edition). Transmission electron microscopy (TEM, TECNAI-10, 80 kV, Philips, Holland) was used to observe ultrastructures of sperm tail. Quantitative realtime-PCR and immunofluorescence staining were performed to detect the expression of DNAH9-mRNA and location of DNAH9-protein. Furthermore, assisted reproductive procedures were applied. Results By WES and Sanger sequencing, compound heterozygous DNAH9 (NM_001372.4) variants were identified in the two individuals with nonsyndromic severe asthenozoospermia (F1 II-1: c.302dupT, p.Leu101fs*47 / c.6956A > G, p.Asp2319Gly; F2 II-1: c.6294 T > A, p.Phe2098Leu / c.10571 T > A, p.Leu3524Gln). Progressive rates less than 1% with normal sperm morphology rates and normal vitality rates were found in both of the two subjects. No respiratory phenotypes, situs inversus or other malformations were found by detailed medical history, physical examination and lung CT scans etc. Moreover, the expression of DNAH9-mRNA was significantly decreased in sperm from F1 II-1. And expression of DNAH9 is lower in sperm tail by immunofluorescence staining in F1 II-1 compared with normal control. Notably, by intracytoplasmic sperm injection (ICSI), F1 II-1 and his partner successfully achieved clinical pregnancy. Conclusions We identified DNAH9 as a novel pathogenic gene for nonsyndromic severe asthenospermia, and ICSI can contribute to favorable pregnancy outcomes for these patients.

scholarly journals Sub-Chronic Effects of Slight PAH- and PCB-Contaminated Mesocosms in Paracentrotus lividus Lmk: A Multi-Endpoint Approach and De Novo Transcriptomic

2021 ◽  
Vol 22 (13) ◽  
pp. 6674
Author(s):  
Luisa Albarano ◽  
Valerio Zupo ◽  
Davide Caramiello ◽  
Maria Toscanesi ◽  
Marco Trifuoggi ◽  
...  
Keyword(s):  
De Novo ◽  
Sea Urchins ◽  
Transcriptome Assembly ◽  
Paracentrotus Lividus ◽  
Marine Biota ◽  
De Novo Transcriptome Assembly ◽  
De Novo Transcriptome ◽  
Limit Values ◽  
The Impact ◽  
Endpoint Approach

Sediment pollution is a major issue in coastal areas, potentially endangering human health and the marine environments. We investigated the short-term sublethal effects of sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) on the sea urchin Paracentrotus lividus for two months. Spiking occurred at concentrations below threshold limit values permitted by the law (TLVPAHs = 900 µg/L, TLVPCBs = 8 µg/L, Legislative Italian Decree 173/2016). A multi-endpoint approach was adopted, considering both adults (mortality, bioaccumulation and gonadal index) and embryos (embryotoxicity, genotoxicity and de novo transcriptome assembly). The slight concentrations of PAHs and PCBs added to the mesocosms were observed to readily compartmentalize in adults, resulting below the detection limits just one week after their addition. Reconstructed sediment and seawater, as negative controls, did not affect sea urchins. PAH- and PCB-spiked mesocosms were observed to impair P. lividus at various endpoints, including bioaccumulation and embryo development (mainly PAHs) and genotoxicity (PAHs and PCBs). In particular, genotoxicity tests revealed that PAHs and PCBs affected the development of P. lividus embryos deriving from exposed adults. Negative effects were also detected by generating a de novo transcriptome assembly and its annotation, as well as by real-time qPCR performed to identify genes differentially expressed in adults exposed to the two contaminants. The effects on sea urchins (both adults and embryos) at background concentrations of PAHs and PCBs below TLV suggest a need for further investigations on the impact of slight concentrations of such contaminants on marine biota.

scholarly journals NEURONAL-GLIAL MEMBRANE CONTACTS DURING PESSIMAL ELECTRICAL STIMULATION

2020 ◽  
Vol 28 (3) ◽  
pp. 35-50
Author(s):  
Oleg S. Sotnikov ◽  
Svetlana S. Sergeeva ◽  
Tat'yana I. Vasyagina
Keyword(s):  
Electrical Stimulation ◽  
Gap Junctions ◽  
De Novo ◽  
Laboratory Rats ◽  
Transmission Electron ◽  
Layer Structures ◽  
Dense Substance ◽  
Membrane Contacts ◽  
Glial Membrane ◽  
Stimulation Of

After the creation of a method for obtaining inter-neuronal gap junctions in a nervous system devoid of glia, it is expedient to reproduce gap neuronal-glial contacts on a model that also contains hybrid neuronal-glial gap junctions, which, as you know, are functionally fundamentally different from inter-neuronal contacts. The experiments were carried out on the truncus sympathicus ganglia of laboratory rats using pessimal electrical stimulation and transmission electron microscopy. Electrical activation of ganglia with a frequency of up to 100 Hz revealed local and widespread variants of various neuronal-glial connections (contacts, bridges), fringed with peri-membrane filamentous proteins. They had a blurred veil that masked two-layer neuro-membranes. Some of the contacts resembled slit or dense 5-layer structures without a visible inter-neuronal slit, but with an extreme decrease in the thickness of the contact slit. The main result of the experiments was the formation, in addition to slotted, multiple septate (ladder) contacts. Relatively independent aggregates of the electron-dense substance of the septa were located inside the intercellular gaps, crossing both adjacent membranes, and, possibly, permeate of them. Near-membrane, poorly outlined pyramid-like protein cones associated with both cell membranes were also formed. Such membranes appeared to be dotted-dashed, that is, not continuous. A significant number of septic contact membranes had endocytic invaginations (invaginations) facing neuroplasm with pyramid-like marginal projections. All reactive altered structures that have arisen de novo are considered by the authors as developed under the influence of frequency electrical stimulation of denaturation and aggregation of intrinsic and perimembrane proteins.

scholarly journals Microtubule assembly by soluble tau impairs vesicle endocytosis and excitatory neurotransmission via dynamin sequestration in Alzheimer's disease synapse model

2021 ◽  
Author(s):  
Tetsuya Hori ◽  
Kohgaku Eguchi ◽  
Han-Ying Wang ◽  
Tomohiro Miyasaka ◽  
Laurent Guillaud ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
De Novo ◽  
Microtubule Assembly ◽  
Pleckstrin Homology Domain ◽  
Endocytic Protein ◽  
Brainstem Slices ◽  
Excitatory Neurotransmission ◽  
Synapse Model ◽  
Activity Dependent

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in brainstem slices. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 μM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin-1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Formation, distribution and dissociation of intercellular junctions in the lens

1987 ◽  
Vol 88 (3) ◽  
pp. 351-359
Author(s):  
W.T. Gruijters ◽  
J. Kistler ◽  
S. Bullivant
Keyword(s):  
Membrane Protein ◽  
Immunofluorescence Microscopy ◽  
De Novo ◽  
Intercellular Junctions ◽  
Polar Regions ◽  
Dynamic Aspect ◽  
Outer Cortex ◽  
Specific Component ◽  
Fibre Bundles ◽  
Fibre Width

A 70,000 Mr membrane protein (MP70) has previously been identified as a specific component of lens intercellular junctions. In this paper we use anti-MP70 immunofluorescence microscopy of dissected fibre bundles to study the formation, distribution and dissociation of junctional plaques in the outer cortex region of the sheep lens. Abundant, small junctional plaques are assembled de novo in the broad sides of the elongating fibres near the equatorial lens periphery. In fully elongated, pole-to-pole fibres, junctional plaques are generally larger, and while dispersed on the broad sides of the fibres in the equatorial lens plane, these junctions line up in the middle of the broad and narrow sides of the fibres in the lens polar regions. This precisely defined positioning is independent of junction size and hence cannot solely be explained by the constraints of fibre width. Junctional plaques fragment to smaller sizes and MP70 is cleaved to MP38 in mature, enucleated fibres located in the deeper portions of the lens outer cortex. These results demonstrate a dynamic aspect of lens intercellular junctions and show that they are positioned in a precise fashion, possibly in association with other membrane or cytoskeletal components.

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