Tatsuo Harumi

Effects of Sperm‐Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water

The effects of sperm‐activating peptide I (SAP‐I: Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K+] sea water were examined. In high [K+] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP‐I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP‐I elevated the sperm cGMP level in 100 mM K+ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX) and SAP‐I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K+ sea water. However, in high [K+] sea water, SAP‐I did not increase the cAMP level even in the presence of IBMX. SAP‐I caused rapid, transient elevation of the intracellular pH and Ca2+ concentration of spermatozoa in normal sea water but not in 100mM K+ sea water. SAP‐I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K+] sea water. These results suggest that the SAP‐I‐induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K+ channels.

Receptors for Sperm‐activating Peptides, SAP‐I and SAP‐IIB, on Spermatozoa of Sea Urchins, Hemicentrotus pulcherrimus and Glyptocidaris crenularis

Sperm‐activating peptide analogues, GYGG‐SAP‐I (Gly‐Tyr‐Gly‐Gly‐Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly) and GYGG‐SAP‐IIB (Gly‐Tyr‐Gly‐Gly‐Lys‐Leu‐Cys‐Pro‐Gly‐Gly‐Asn‐Cys‐Val) which exibit almost the same respiration‐stimulating activity as the respective original peptide were chemically synthesized, radiolabeled with Na1251, and used for experiments of binding and cross‐linking to Hemicentrotus pulcherrimus or Glyptocidaris crenularis spermatozoa. In competitive binding, SAP‐I caused 50% decrease in 125l‐GYGG‐SAP‐l binding to intact spermatozoa, sperm heads and sperm tails of H. pulcherrimus at the concentrations of 282 nM, 3 nM and 141 nM, respectively. The incubations of H. pulcherrimus sperm tails with 125l‐GYGG‐SAP‐I and the chemical cross‐linking reagent, disuccinimidyl suberate, resulted in the appearance of two radiolabeled bands with apparent molecular masses of 71 kDa and 63 kDa (determined by sodium dodecyl sulfate‐gel electrophoresis under reducing conditions). 125l‐GYGG‐SAP‐IIB bound almost equally to sperm heads and sperm tails of G. crenularis, and was cross‐linked to 172 kDa, 62 kDa and 58 kDa proteins in sperm heads, and 157 kDa and 62 kDa proteins in sperm tails with disuccinimidyl suberate.

Purification and characterization of sperm creatine kinase and guanylate cyclase of the sea urchin Hemicentrotus pulcherrimus

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA‐Sepharose column connected to a Concanavalin A‐Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS‐PAGE. The specific activity of the purified enzyme was 8.25 μmol of cGMP formed/min/mg protein. Sperm‐activating peptide I (SAP‐I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co‐purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP‐I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.

The influence of an egg-associated peptide on energy metabolism in sea-urchin spermatozoa: the peptide stimulates preferential hydrolysis of phosphatidylcholine and oxidation of fatty acid

A study was made of the effects of a sperm-activating peptide (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. The swimming activity and respiratory rate in slightly acidic seawater (pH 6.6) have been shown to be somewhat less than in normal seawater (pH 8.2). Little change occurred in sperm lipid levels during incubation in seawater at pH 6.6. The addition of SAP-I to seawater at pH 6.6 enhanced the consumption of endogenous phosphatidylcholine (PC), with no change in the levels of other lipids. SAP-I also caused increase in 14CO2 production from exogenous [1-14C]oleic acid following incubation of spermatozoa at pH 6.6. However, the stimulated levels of PC consumption and fatty acid oxidation with SAP-I at pH 6.6 did not exceed those at pH 8.2. At pH 8.2, SAP-I had no effect on PC metabolism. Activities of phospholipase A2 and fatty acid oxidation were markedly influenced by pH and increased at pH exceeding 7. SAP-I is thus concluded to stimulate sea-urchin sperm energy metabolism which depends on the oxidation of endogenous PC. It follows from these results that PC metabolism is activated following increase in the intracellular pH of spermatozoa.

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm‐activating peptide I (SAP‐I: Gly‐Phe‐Asp‐Leu‐Asn‐Gly‐Gly‐Gly‐Val‐Gly), on the intracellular pH (pHi) and Ca2+ ([Ca2+]i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus. FSG and/or SAP‐I induced elevations of [Ca2+]; and pHi in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca2+]i or pHi of spermatozoa treated with FSG, but addition or FSG after SAP‐I or of SAP‐I after FSG induced further increases of [Ca2+]i and pHi, At pH 6.6, FSG and/or SAP‐I did not induce significant elevation of the [Ca2+]i, although SAP‐I elevated the pHi, its half‐maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl‐ammonium, a voltage‐sensitive K+‐channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca2+]i and pHi by FSG, but did not affect those by SAP‐I. These results suggest that FSG and SAP‐I activate different Ca2+ and H+ transport systems.