Sperm surface proteases in ascidian fertilization.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization.