Evidence for cell surface and internal phospholipase activity in ascidian eggs

Area of Science:

• Reproductive biology
• Cell biology
• Biochemistry

Background:

• Fertilization in ascidian eggs triggers cortical contractions and the release of a cell surface glycosidase.
• This glycosidase plays a role in preventing polyspermy (fertilization by multiple sperm).
• Intracellular calcium levels are known to increase upon fertilization, influencing these events.

Purpose of the Study:

• To investigate the role of phospholipase C (PLC) in the release of cell surface glycosidase during ascidian fertilization.
• To determine the localization of PLC activity within ascidian eggs.

Main Methods:

• Assessing glycosidase release triggered by fertilization, calcium ionophores, and exogenous PLC.
• Using a specific PLC substrate (4-methylumbelliferyl-phospho-choline) to detect enzymatic activity.
• Employing ion affinity chromatography to separate different phospholipase forms.
• Utilizing biotinylation of surface proteins to determine the cell surface localization of PLC activity.

Main Results:

• PLC inhibitor D609 blocked glycosidase release, indicating PLC's involvement.
• The fluorogenic substrate was cleaved specifically by PLC and did not enter intact eggs.
• Ionomycin stimulated both glycosidase release and PLC activity.
• Ascidian egg extracts revealed two forms of phospholipase activity (PL1 and PL2).
• Half of the total PLC activity was found to be associated with the cell surface.

Conclusions:

• Phospholipase C (PLC) activity is essential for the release of cell surface glycosidase in ascidian eggs, contributing to the block to polyspermy.
• Ascidian eggs possess both intracellular and cell surface-associated PLC activity.