Japonicus Eggs Research Articles

Analysis of the breakdown of cortical granules in echinoderm eggs by microinjection of second messengers.

Breakdown of cortical granules was investigated by microinjecting Ca-EGTA buffers at various concentrations of Ca2+, inositol 1, 4, 5- trisphosphate (IP3), guanosine triphosphate (GTP) and guanosine 5' -O- thiotriphosphate (GTPγS) into echinoderm (Clypeaster japonicus, Temnopleurus hardwicki, and Scaphechinus mirabilis) eggs using a video microscope. The breakdown started in the region near the injection site and then propagated over the entire surface of the egg. The cortical granules did not synchronously break down side by side, but instead underwent random breakdown, in the same manner as that upon fertilization. The reaction times of the breakdown of the cortical granule after the injection of Ca-EGTA buffers, IP3, and GTPγS were 0.4 sec, 1.0 sec, and 2.7 sec, respectively, in C. japonicus eggs, and were in the same order in T. hardwicki and S. mirabilis eggs. It is inferred from this order of reaction times that GTP-binding proteins, IP3 and Ca2+ act in this sequence upon fertilization in echinoderm eggs.

Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM- or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC- or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 microM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.

Actin filament organization in the sand dollar egg cortex

The organization of actin filaments in the sand dollar egg cortex was studied by fluorescence microscopy using an F-actin specific stain, NBD-phallacidin (NBD-Ph). Cortices were prepared from Clypeaster japonicus eggs which had been stuck down onto polycation-coated glass surfaces and sheared with a stream of a medium capable of preserving actin-containing structures. Cortices from unfertilized eggs showed weak fluorescence with NBD-Ph. Immediately after fertilization, however, isolated cortices were brightly fluorescent. Fluorescent spikes which seemed to correspond to actin filament bundles in microvilli were prominent structures of cortices isolated before and at the streak stage. At anaphase, a fluorescent meshwork which seemed to consist of actin filament bundles was observed all over the isolated cortex. At telophase, the contractile ring was observed as brightly fluorescent oriented fibers in the equatorial cortex. The fluorescent meshwork still remained in the rest of the cortex. Soon after cleavage, the fluorescent meshwork became obscure and fluorescent spikes appeared again. When whole eggs were stained with NBD-ph, the fibrous structures were not identifiable, although the changes in the fluorescence intensity of the cortical layer were consistent with those in isolated cortices. The process of formation of the contractile ring is discussed.