Phospholipase C-dependent Ca 2 + release by worm and mammal sperm factors q Kethurah P. Howell, a,b Aurelia Skipwith, a,b Antony Galione, b,c and William R. Eckberg a,b, * a Department of Biology, Howard University, 415 College Street, NW, Washington, DC 20059-0001, USA b Marine Biological Laboratory, Woods Hole, MA 02543, USA c Department of Pharmacology, University of Oxford, Mansfield Rd., Oxford OX1 3QT, UK Received 30 May 2003 Abstract Egg activation in all animals evidently requires the synthesis of inositol 1,4,5-trisphosphate (InsP 3 ) from phosphatidylinositol 4,5- bisphosphate (PIP 2 ) by phospholipase C (PLC). Depending on the organism, InsP 3 elicits either calcium oscillations or a single wave, which in turn initiates development. A soluble component in boar sperm that activates mammalian eggs has been suggested to be a PLC isoform. We tested this hypothesis in vitro using egg microsomes of Chaetopterus. Boar sperm factor elicited Ca 2þ release from the microsomes by an InsP 3 -dependent mechanism. The PLC inhibitor U-73122, but not its inactive analog U-73343, blocked the response to sperm factor but not to InsP 3 . U-73122 also inhibited the activation of fertilized and parthenogenetic eggs. Chaetopterus sperm also contained a similar activity. These results strongly support the hypothesis that sperm PLCs are ubiquitous mediators of egg activation at fertilization. Ó 2003 Elsevier Science (USA). All rights reserved. Egg activation at fertilization results from one or more waves of intracellular Ca 2þ release [1–3]. This re- lease originates from intracellular calcium stores [4–6] and is elicited by one or more intracellular second messengers. These include inositol 1,4,5-trisphosphate (InsP 3 ), cyclic adenosine diphosphate ribose (cADPR), and nicotinate adenine dinucleotide phosphate (NA- ADP). The receptors for InsP 3 and cADPR are on the endoplasmic reticulum (ER) [7]; those for NAADP are on lysosomal-related organelles [8]. Of these three, cADPR and NAADP release Ca 2þ in sea urchin eggs and homogenates [9–12], but not those of most other organisms [2]. However, all animal eggs apparently utilize InsP 3 in their Ca 2þ release at fertilization [13–17]. The biochemical processes leading to InsP 3 synthesis and Ca 2þ release at fertilization have been controversial. Two general classes of hypotheses, involving either sperm–egg contact or fusion, differ in that contact-based hypotheses suggest that sperm activate eggs by binding to receptors and activating intracellular signal trans- duction systems similar to those in somatic cells [18], whereas fusion-based hypotheses suggest that sperm introduce some soluble substance into the egg that di- rectly activates some step in the signal transduction process [11,18]. While there is evidence to suggest that sperm can activate eggs of at least one species by contact [19], more recent research has been directed at soluble sperm fac- tors that could be introduced into the egg after fusion. The sperm factor hypothesis is based on the observa- tions that microinjection of sperm [20] or soluble ex- tracts from sperm but not other tissues can elicit Ca 2þ responses characteristic of fertilization and activate eggs [21–23]. Such activity is apparently widespread in the animal kingdom, being found in vertebrates [22,24,25], non-vertebrate chordates [26], and nemertean worms [21]. Since mammalian sperm have phospholipase C (PLC) activity that is capable of synthesizing InsP 3 [15], it was Biochemical and Biophysical Research Communications 307 (2003) 47–51 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: CaFASW, calcium-free artificial seawater; cADPR, cyclic adenosine diphosphate ribose; InsP 3 , inositol (1,4,5)- trisphosphate; NAADP, nicotinate adenine dinucleotide phosphate; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C. * Corresponding author. Fax: 1-202-806-4564. E-mail address: weckberg@howard.edu (W.R. Eckberg). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01120-3