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

Abstract

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 Clypeuster juponicus 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 pM), 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 intracelluiar 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.

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

“…First of all, we observed the development of the sperm‐incorporated egg. We confirmed the results of previous reports (Hamaguchi & Hiramoto 1981; Hamaguchi & Mabuchi 1988; Mohri & Hamaguchi 1991): sperm can enter eggs injected with EGTA or BAPTA and the sperm head becomes a male pronucleus, but the sperm aster does not form. The exocytosis of cortical granules and the formation of the fertilization envelope do not occur and consequently these eggs are liable to be polyspermic.…”

“…This suppression is presumed to be due to the lack of MTOC, but no experi‐mental evidence has been reported. It has been reported previously that sperm aster formation and further development are inhibited when the transient increase in Ca i is blocked by injection of ethyleneglycol‐bis(β‐aminoethyl ether)‐ N,N,N′,N′ ‐tetraacetic acid (EGTA; Hamaguchi & Hiramoto 1981; Hamaguchi & Mabuchi 1988; Mohri & Hamaguchi 1991). However, in these studies, pH i was not measured.…”

Simultaneous investigation of intracellular Ca2+ increase and morphological events upon fertilization in the sand dollar egg.

“…The top micrographs show that the un fertilized egg exhibited both green fluorescence increase and FE elevation when [Ca2+] in the egg was raised up to 2.1 juM by injecting the calcium buffer solution following the injection of Fluo-3. On the other hand, EGTAsolution may lower [Ca2+] in the egg cytoplasm by chelating Ca2+ when injected into sea urchin eggs (5,6). Soon after two of three un fertilized eggs were fertilized following the injection of Fluo-3, the green fluorescence increased and FE elevation was induced (e and f in the bottom micrographs of Fig.…”

Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice

“…5, incorporation of exogenous nonmuscle actin into the microfilament of the mouse egg is obvious. In sea urchin eggs, microinjected actins distributed evenly in the cytoplasm and accumulated first around the sperm (Hamaguchi and Mabuchi, 1988). Microinjected actins accumulate near the egg cortex, especially adjacent to the meiotic spindle.…”