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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 EmbryosTo 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.
Document ID
19970004941
Acquisition Source
Kennedy Space Center
Document Type
Conference Paper
Authors
Chakrabarti, Amitabha (Wisconsin Univ. Madison, WI United States)
Stoecker, Andrew (Wisconsin Univ. Madison, WI United States)
Schatten, Heide (Wisconsin Univ. Madison, WI United States)
Date Acquired
September 6, 2013
Publication Date
January 1, 1995
Publication Information
Publisher: The American Inst. of Aeronautics and Astronautics
Subject Category
Life Sciences (General)
Report/Patent Number
NASA-CR-202584
NAS 1.26:202584
AIAA Paper 95-1059
Meeting Information
Meeting: Life Sciences and Space Medicine
Location: Houston, TX
Country: United States
Start Date: April 3, 1995
End Date: April 5, 1995
Sponsors: American Inst. of Aeronautics and Astronautics
Accession Number
97N12941
Funding Number(s)
CONTRACT_GRANT: NAG10-64
Distribution Limits
Public
Copyright
Public Use Permitted.

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