A species‐specific sperm factor dispersing the jelly coat of the egg of the sea urchin, Anthocidaris crassispina

Abstract

A species-specific factor capable of dispersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassispina. It does not exert its effect on the vitelline layer. The purification has been accomplished by a four-step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL-4B, ion-exchange column chromatography on DEAE-cellulose, and affinity column chromatography on heparin-Sepharose CL-6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 0-rnercaptoethanol, estimated molecular weight being about 140,000.The jelly dispersion by the present factor is activated by CaCI2, and inhibited by KCI, MnCI2, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose-6-sulfate. Inorganic sulfates such as (NH&S04 and Na2S04 have no effect on jelly dispersion. This factor is heat-labile, losing its activity in 30 min at 50°C.The present factor is found also in the seminal plasma, and released from sperm themselves by treatment with Triton X-100. These results suggest that this factor is loosely bound to the sperm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly dispersing factor.Only trypsin and or-chymotrypsin among commercial enzymes tested disperse the jelly coat, but neither activity is detectable in our preparation. The jelly-dispersing activity is inhibited neither by trypsin inhibitors such as N-a-p-tosyl-L-lysine-chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L-I-tosylamide-2-phenyl-ethylchloromethyl ketone and chymostatin. Participation of trypsin-like and chymotrypsin-like enzymes in jelly dispersion seems unlikely.

Sperm Penetration of the Vitelline Envelope of Sicyonia ingentis Eggs is Mediated by a Trypsin‐like Lysin of Acrosomal Vesicle Origin

“…In another ascidian, Ciona intestinalis, belonging to a different family than H. roretzi, only a chymotrypsin-like enzyme is involved in sperm penetration through the egg's vitelline coat (34,41). Trypsin and chymotrypsinlike enzymes have been purified, characterized, and their location demonstrated at the finestructural level in sea urchins (12,28,55,57). Yamada and Aketa (54, 56) and Green and Summers (1 3) have demonstrated that these enzymes promote jelly coat dispersal and lysis of the VE.…”

“…Described egg coat lysins can be classified into one of two general categories, enzymatic and stoichiometric. Enzymatic lysins, including acrosin and other trypsin-like and chymotrypsin-like proteases, are reported from mammals (42), anuran amphibians (58), ascidians (23,34), and echinoderms (12,13,28,29,54,55,56,57). Non-enzymatic or stoichiometric lysins are found, to date, in only one group, the molluscs (17, 18,30).…”