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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1985

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

Schatten, G.↗
NASA NTRS1986

Motility and centrosomal organization during sea urchin and mouse fertilization

It is noted that microfilaments are essential for incorporation of sperm in sea urchins and for pronuclear apposition in mice. The ability of sea urchin sperm to fertilize eggs is lowered by latrunculin, giving evidence that acrosomal microfilaments are of importance to the process of fertilization. Due to the uncertainty regarding the presence of microfilaments in various mammalian sperm, it is interesting that latrunculin does not noticeably affect the ability of mouse sperm to fertilize oocytes. The movements of the sperm and egg nuclei at the time of sea urchin fertilization are dependent on microtubules arranged into a radial monastral array (the sperm aster). In the mouse egg, microtubule activity is also required during pronuclear apposition, but they are arranged by a number of egg cytoplasmic sites. Results of the investigations show that both microtubules and microfilaments are necessary for the successful completion of fertilization in both mice and sea urchins, but at different stages. Also, it is demonstrated that centrosomes are contributed by the sperm in the process of sea urchin fertilization, but in mammals they may be inherited maternally.

Schatten, Heide↗
NASA NTRS2000

Spatial expression of Hox cluster genes in the ontogeny of a sea urchin

The Hox cluster of the sea urchin Strongylocentrous purpuratus contains ten genes in a 500 kb span of the genome. Only two of these genes are expressed during embryogenesis, while all of eight genes tested are expressed during development of the adult body plan in the larval stage. We report the spatial expression during larval development of the five 'posterior' genes of the cluster: SpHox7, SpHox8, SpHox9/10, SpHox11/13a and SpHox11/13b. The five genes exhibit a dynamic, largely mesodermal program of expression. Only SpHox7 displays extensive expression within the pentameral rudiment itself. A spatially sequential and colinear arrangement of expression domains is found in the somatocoels, the paired posterior mesodermal structures that will become the adult perivisceral coeloms. No such sequential expression pattern is observed in endodermal, epidermal or neural tissues of either the larva or the presumptive juvenile sea urchin. The spatial expression patterns of the Hox genes illuminate the evolutionary process by which the pentameral echinoderm body plan emerged from a bilateral ancestor.

Non-NASA Center↗
NASA NTRS1992

Activation of maternal centrosomes in unfertilized sea urchin eggs

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.

Non-NASA Center↗
NASA NTRS1998

Centrosome structure and function is altered by chloral hydrate and diazepam during the first reproductive cell cycles in sea urchin eggs

This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their ability to reform normal mitotic figures. These results indicate that chloral hydrate and diazepam affect centrosome structure which results in the inability to reform normal microtubule formations and causes abnormal fertilization and mitosis.

Non-NASA Center↗
NASA NTRS2003

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗
NASA NTRS1996

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

NASA Discipline Musculoskeletal↗
NASA NTRS1999

Microgravity Effecs During Fertilization, Cell Division, Development, and Calcium Metabolism in Sea Urchins

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. For long-term exposure to space it is crucial to understand the underlying mechanisms for altered physiological functions. Fundamental occurrences in cell biology which are likely to depend on gravity include cytoskeletal dynamics, chromatin and centrosome cycling, and ion immobilization. These events can be studied during fertilization and embryogenesis within invertebrate systems. We have chosen the sea urchin system to study the effects of microgravity on cytoskeletal processes and calcium metabolism during fertilization, cell division, development, and embryogenesis. Experiments during an aircraft parabolic flight (KC-135) demonstrated: (1) the viability of sea urchin eggs prior to fertilization, (2) the suitability of our specimen containment system, (3) the feasibility of fertilization in a reduced gravity environment (which was achieved during 25 seconds of reduced gravity under parabolic flight conditions). Two newly developed pieces of spaceflight hardware made further investigations possible on a spaceflight (STS-77); (1) the Aquatic Research Facility (ARF), and (2) the Fertilization Syringe Unit (FSU). The Canadian Space Agency developed ARF to conduct aquatic spaceflight experiments requiring controlled conditions of temperature, humidity, illumination, and fixation at predetermined time points. It contained a control centrifuge which simulated the 1 g environment of earth during spaceflight. The FSU was developed at the Kennedy Space Center (KSC) by the Bionetics Corporation specifically to enable the crew to perform sea urchin fertilization operations in space.

Schatten, Heide↗
NASA NTRS1986

Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

Schatten, Heide↗
NASA NTRS1992

Microgravity effects of sea urchin fertilization and development

Gravity has been a pervasive influence on all living systems and there is convincing evidence to suggest that it alters fertilization and embryogenesis in several developmental systems. Notwithstanding the global importance of gravity on development, it has only been recently possible to begin to design experiments which might directly investigate the specific effects of this vector. The goal of this research program is to explore and understand the effects of gravity on fertilization and early development using sea urchins as a model system. Sea urchin development has several advantages for this project including the feasibility of maintaining and manipulating these cells during spaceflight, the high percentage of normal fertilization and early development, and the abundant knowledge about molecular, biochemical, and cellular events during embryogenesis which permits detailed insights into the mechanism by which gravity might interfere with development. Furthermore, skeletal calcium is deposited into the embryonic spicules within a day of fertilization permitting studies of the effects of gravity on bone calcium deposition.

Steffen, S.↗
NASA NTRS1998

Chloral hydrate alters the organization of the ciliary basal apparatus and cell organelles in sea urchin embryos

The mitotic inhibitor, chloral hydrate, induces ciliary loss in the early embryo phase of Lytechinus pictus. It causes a breakdown of cilia at the junction of the cilium and the basal body known as the basal plate. This leaves the plasma membrane temporarily unsealed. The basal apparatus accessory structures, consisting of the basal body, basal foot, basal foot cap, striated side arm, and striated rootlet, are either misaligned or disintegrated by treatment with chloral hydrate. Furthermore, microtubules which are associated with the basal apparatus are disassembled. Mitochondria accumulate at the base of cilia - underneath the plasma membrane - and show alterations in their structural organization. The accumulation of mitochondria is observed in 40% of all electron micrograph sections while 60% show the areas mostly devoid of mitochondria. The microvilli surrounding a cilium and striated rootlet remain intact in the presence of chloral hydrate. These results suggest that deciliation in early sea urchin embryos by chloral hydrate is caused by combined effects on the ciliary membrane and on microtubules in the cilia. Furthermore, it is suggested that chloral hydrate can serve as a tool to explore the cytoskeletal mechanisms that are involved in cilia motility in the developing sea urchin embryo.

Non-NASA Center↗
NASA NTRS1999

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions. Copyright 1999 Academic Press.

STS-77 Shuttle Project↗
NASA NTRS1995

Modification of Experimental Protocols for a Space Shuttle Flight and Applications for the Analysis of Cytoskeletal Structures During Fertilization, Cell Division , and Development in Sea Urchin Embryos

To explore the role of microgravity on cytoskeletal organization and skeletal calcium deposition during fertilization, cell division, and early development, the sea urchin was chosen as a model developmental system. Methods were developed to employ light, immunofluorescence, and electron microscopy on cultures being prepared for flight on the Space Shuttle. For analysis of microfilaments, microtubules, centrosomes, and calcium-requiring events, our standard laboratory protocols had to be modified substantially for experimentation on the Space Shuttle. All manipulations were carried out in a closed culture chamber containing 35 ml artificial sea water as a culture fluid. Unfertilized eggs stored for 24 hours in these chambers were fertilized with sperm diluted in sea water and fixed with concentrated fixatives for final fixation in formaldehyde, taxol, EGTA, and MgCl2(exp -6)H2O for 1 cell to 16 cell stages to preserve cytoskeletal structures for simultaneous analysis with light, immunofluorescence, and electron microscopy, and 1.5 percent glutaraldehyde and 0.4 percent formaldehyde for blastula and plueus stages. The fixed samples wre maintained in chambers without degradation for up to two weeks after which the specimens were processed and analyzed with routine methods. Since complex manipulations are not possible in the closed chambers, the fertilization coat was removed from fixation using 0.5 percent freshly prepared sodium thioglycolate solution at pH 10.0 which provided reliable immunofluorescence staining for microtubules. Sperm/egg fusion, mitosis, cytokinesis, and calcium deposition during spicule formatin in early embryogenesis were found to be without artificial alterations when compared to cells fixed fresh and processed with conventional methods.

Chakrabarti, Amitabha↗
NASA NTRS2002

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages. (c) 2002 Elsevier Science (USA).

Non-NASA Center↗
NASA NTRS1971

Sea urchin egg fertilization and development

The effects of subgravity (much less than unit gravity) on fertilization, cell division, differentiation, and growth of a relatively simple biological system (eggs of the sea urchin Arbacia punctulata) were considered. The experiment was flown on Gemini 3 and recovered as scheduled. However, the experiment objectives were not achieved, primarily for mechanical reasons.

Young, R. S.↗