Vitelline coat lysins from molluscan sperm and their use in microinjection

Microinjection through the tough vitelline coats of oocytes from three molluscan species (Haliotis rufescens, Norrisia norrisi, and Astrea undosa) was facilitated by the local appli- cation of sperm vitelline coat lysin with a micropipet. Extracts containing lysin were the 12,OOOg supernates of frozen-thawed sperm. In H. rufescens and N. norrisi, lysin extracts dissolved a small hole through the vitelline coat, whereas in A. undosa, the lysin created a pathway for mi- croinjection by softening the vitelline coat without dissolving it. Similar effects were observed when oocytes were suspended in lysin extracts: Vitelline coats dissolved completely in H. rufescens (previously reported by Lewis et al. (19821, Dev. Biol. 92:227-239) and N. norrisi, but only soft- ened and swelled in A. undosa (although they could then be removed by mechanical agitation). The lysin extracts were highly species specific and had no visible effects on heterologous vitelline coats even at concentrations higher than those required to dissolve/soften homologous coats. Evi- dence that N. norrisi lysin acts by a non-enzymatic mechanism was provided by the observation that mobilities of radioactive bands in sodium dodecyl sulfate polyacrylamide gels were unchanged following dissolution of lZ5I-labeled vitelline coats. Electron micrographs of the previously undescribed sperm from N. norrisi and A. undosa are also presented. o 1995 \ViIey-Liss, Inc. In many animal species, the exposure of lytic agents, called lysins, during the acrosome reac- tion enables sperm to penetrate through the ex- ternal coat of the egg and fuse with the egg plasma membrane (reviewed by Hoshi, '85). Among inver- tebrates, most information about lysins comes from the molluscs (Hoshi, '85). Sperm lysins that dissolve egg vitelline coats (VCs) have been ob- tained from the giant keyhole limpet Megathura crenulata (Tyler, '391, the mussells MytiZus edulis and californianus (Berg, '501, various snails (Tegula species, Haino, '71; ?2lrbo cornutus, Ogawa and Haino-Fukushima, '84), and abalone (Haliotis corrugata, cracheroderi, discus and rufescens: Tyler, '39; Lewis et al., '82; Haino-Fukushima and Usui, '86; Vacquier et al., '90). In Tegula (Haino- Fukushima, ,741, Haliotis (Lewis et al., '82), and %rho (Ogawa and Haino-Fukushima, '84), the mechanism of action of lysin is non-enzymatic: a stoichiometric combination of lysin molecules with yet-to-be-identified VC components causes the dis- solution of the VC with no evidence of covalent bond breakage. When we initiated experiments involving the microinjection of abalone oocytes, we found that it was difficult to penetrate the tough VC with- out damaging or killing the oocyte. Removal of