Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.
The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.
A complementary DNA clone (2.3 kb) that encodes the egg peptide speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) has been isolated from an ovary cDNA library of the sea urchin. Stronglyocentrotus purpuratus. The DNA sequence predicts an open reading frame of 296 amino acids. The likely site of initiation, however, is a downstream in-frame translation initiation codon that would result in a polypeptide of 260 amino acids containing 10 decapeptides, each separated by a single lysine residue. Four of the peptides are speract, and six have the predicted structures of Gly-Phe-Ala-Leu-Gly-Gly-Gly-Gly-Val-Gly (occurs twice), Gly-Phe-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly, Gly-Phe-Ser-Leu-Thr-Gly-Gly-Gly-Val-Gly, Gly-Thr-Met-Pro-Thr-Gly-Ala-Gly-Val-Asp, and Ile-Asp-His-Asp-Thr-Leu-Ala-Ser-Val-Ser. The isolated cDNA insert hybridized to two species of ovarian mRNA (1.2 and 2.3 kb) obtained from species known to produce speract or speract-like peptides, but failed to hybridize to RNA from other species. Subsequently, a second ovarian cDNA clone (1.2 kb) was isolated and sequenced; this clone contained two additional potential decapeptides: Ser-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly and Ser-Thr-Met-Pro-Thr-Gly-Ala-Gly-Val-Asp. The various speract and speract-like peptides found in egg-conditioned media, therefore, reflect, in part, variable structures within a single copy of mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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