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

X-Ray Microanalysis of Calcium Containing Organelles in Resin Embedded Tissue

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites : calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation. Resin embedding is less demanding than cryomicrotomy and gives better images : it can be used after cryosubstitution in the presence of oxalic acid. This technique was tested, and applied to several cell types

Effects of Ionomycin on Egg Activation and Early Development in Starfish

Ionomycin is a Ca2+-selective ionophore that is widely used to increase intracellular Ca2+ levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca2+ levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca2+ increase. The ionomycin-induced Ca2+ rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca2+ response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation

Effects of progesterone on human spermatozoa prepared for in‐vitro fertilization

International audienceSummary Progesterone has been tested in vitro with human spermatozoa to verify its physiological effects and its possible therapeutic use in cases of male infertility. Progesterone induced a rapid, dose‐dependent influx of calcium in capacitated and non‐capacitated spermatozoa with a half‐maximally effective dose of 30 nM. The agonist, 19‐nortestosterone, was much less potent that progesterone itself. Progesterone‐induced calcium influx was not inhibited by a similar concentration (0.1 μg/ml) of RU 486, a classical progesterone antagonist. The increase in intracytoplasmic calcium levels was unable to induce the acrosome reaction (AR) even after incubation for 5 h, when this was evaluated by double staining, using a monoclonal antibody GB24 raised against the inner acrosome membrane and ethidium homodimer as a vital probe. However, after incubation for 5 h, progesterone was able to enhance the tyrosine phosphorylation of a 95 kD sperm protein, which is phosphorylated progressively during capacitation in well‐defined culture media. Incubation of spermatozoa with 1 or 10 μg/ml progesterone for 3 or 30 min did not induce major modifications of hyperactivated movement when analysed by computer‐assisted semen analysis. Progesterone secreted by cumulus cells may physiologically increase sperm intracytoplasmic free calcium during capacitation. This priming effect may facilitate the acrosome reaction, induced by binding to the zona pellucida, without enhancing spontaneous acrosome reaction prematurely. It therefore seems useful to propose progesterone as a means of accelerating capacitation during in vitro fertilization in cases of male infertility