Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane, the other involving the follicle cells

Abstract

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N‐acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N‐acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137‐143, 1997. © 1997 Wiley‐Liss, Inc.

Localization of Ubiquitin C-Terminal Hydrolase L1 in Mouse Ova and Its Function in the Plasma Membrane to Block Polyspermy

“…The mechanism that prevents polyspermy is classically illustrated by the zona reaction (cortical reaction) and subsequent plasma membrane block. 33,34 After penetration by sperm, the zona reaction occurs, and cortical contents are extruded into the perivitelline space, thereby preventing polyspermic penetration by hardening of the zona pellucida. 35 Plasma membrane block is assumed to result from changes (eg, the destruction of sperm receptors in the membrane) that preclude sperm adherence.…”

Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

“…␤-hexosaminidase was suggested to be primarily released from the egg cell surface to which it was bound via a glycosylphosphatidylinositol anchor (Lambert & Goode 1992). However, sperm-triggered release of a lipid-linked ␤-hexosaminidase from the follicle cells is now suggested to be involved in the early block to polyspermy (Lambert et al 1997).…”

“…Because glycosidases usually have a low activity at neutral and alkaline conditions, a transitory stable lectin-like enzyme-substrate binding would be established in sea water (Hoshi 1984;Honegger 1986;Lambert 1989b;Godknecht & Honegger 1991;Godknecht & Honegger 1995). The currently favored model of ascidian fertilization claims that sperm ␤ -hexosaminidase participates in transient spermegg binding and that ␤ -hexosaminidase released from the egg contributes to the block against polyspermy (Lambert 1989a,b;Lambert et al . 1997).…”

The ascidian egg envelope in fertilization: structural and molecular features

“…FC have also been proposed to participate in a physical (De Santis et al, 1980) or chemical block to polyspermy (Lambert et al, 1997). Whereas a polyspermy block caused by a purely physical barrier is rather unlikely and not supported by in-depth studies, increasing evidence for a block due to a modification of sperm binding sites upon fertilization has been accumulated.…”

“…This block would precede an electrically mediated block at the egg membrane which inhibits fusion with supernumerary sperm (Goudeau et al, 1994, Lambert et al, 1997. The hypothesis of a glycosidase mediated block to polyspermy was quickly presented as a fact although fundamental questions remained to be answered.…”