Carroll D J, Jaffe L A
Dept. of Physiology, University of Connecticut Health Center, Farmington 06032, USA.
Dev Biol. 1995 Aug;170(2):690-700. doi: 10.1006/dbio.1995.1247.
Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (congruent to 30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodeoxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 nM) such that Ca influx would be reduce by > 10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.
将蛋白酶应用于小海星（Asterina miniata）的卵，会引发几种类似于受精时所见的反应。在接触胰蛋白酶、胰凝乳蛋白酶或链霉蛋白酶后约1分钟内，会发生皮质颗粒胞吐作用和受精膜升高；蛋白酶抑制剂可阻止这些反应。激肽释放酶会引起皮质颗粒胞吐作用和受精膜升高，但这种反应需要更多时间（约30分钟）。对重组胰蛋白酶有反应也会出现胞吐作用，但对无蛋白水解活性的点突变胰蛋白酶则无反应。通过荧光染料FM 1 - 43测量，胞吐作用的程度与受精时所见相似。除了引起胞吐作用外，应用胰蛋白酶、胰凝乳蛋白酶或链霉蛋白酶还会导致细胞内游离钙增加（通过钙绿葡聚糖检测），以及刺激DNA合成（通过溴脱氧尿苷掺入检测）。当在游离钙降低至低水平（40 - 70 nM）的人工海水中应用胰蛋白酶或胰凝乳蛋白酶时，也会发生胞吐作用，这样钙内流将减少>10,000倍；这表明蛋白酶并非通过损伤卵并导致外部钙泄漏到细胞质中起作用。这些发现表明，存在一种细胞外暴露的蛋白质，当被蛋白水解时可诱导类似受精的反应；这种蛋白质可能是一种受体，在受精时转导来自精子的信号以启动卵激活。
