Masatoshi Mita

Involvement of G-proteins and adenylate cyclase in the action of gonad-stimulating substance on starfish ovarian follicle cells☆

Gonad-stimulating substance (GSS) secreted from radial nerves induces meiotic maturation of starfish oocytes by stimulating production of 1-methyladenine (1-MeAde) in ovarian follicle cells. We have previously shown that cAMP mediates the action of GSS on 1-MeAde synthesis by starfish ovarian follicle cells. The present study examines the possible involvement of guanine nucleotide-binding regulatory proteins (G-proteins) and adenylate cyclase in the action of GSS on 1-MeAde production by starfish (Asterina pectinifera) follicle cells. GSS slightly stimulated adenylate cyclase activity in crude membrane preparations of follicle cells. GTP markedly enhanced this action of GSS in a dose-dependent manner. Nonhydrolyzable GTP analogs such as guanosine 5-O-(3-thiotriphosphate) and 5-guanylylimidodiphosphate, NaF, and forskolin also stimulated adenylate cyclase activity. In addition, chorela toxin (CT) stimulated adenylate cyclase activity in membrane preparations in the presence of NAD and GTP. Unlike adenylate cyclase, phosphodiesterase activity was not influenced by GSS. When crude membranes of follicle cells were incubated with [alpha-32P]NAD in the presence of CT and pertussis toxin, 45-kDa and 41-kDa proteins were ADP-ribosylated, respectively, suggesting the presence of two types (stimulatory and inhibitory) of G-proteins. It is concluded that G-proteins and adenylate cyclase play an important role in the action of GSS on 1-MeAde production by starfish ovarian follicle cells.

Regulatory functions of cyclic adenosine 3',5'-monophosphate in 1-methyladenine production by starfish follicle cells

The biosynthesis of 1-methyladenine (1-MeAde) in follicle cells of the starfish, Asterina pectinifera, occurred in response to a gonad-stimulating substance (GSS). Simultaneously with 1-MeAde production, the intracellular cAMP level immediately increased following the administration of GSS. This level in follicle cells markedly depended on GSS concentration. Although 1-MeAde production was also induced by 1-methyladenosine, it caused no increase in cAMP content. It thus appears that the effect of GSS on starfish follicle cells results in the receptor-mediated formation of cAMP.

Mediation of cyclic adenosine 3′,5′-monophosphate in 1-methyladenine production by starfish ovarian follicle cells

Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). It has also been reported that concanavalin A (Con A) and two serine proteolytic enzymes (trypsin and Pronase) can stimulate 1-MeAde production. This study was undertaken to determine if 1-MeAde production induced by these compounds is mediated through elevation of cAMP in starfish (Asterina pectinifera) follicle cells. GSS at a concentration of 0.1 mg/ml significantly stimulated 1-MeAde accumulation in extracellular medium after 1-2 hr of follicle cell incubations. GSS also caused a four- to fivefold increase in intracellular levels of cAMP. The continuous presence of GSS was required for the maintenance of elevated levels of cAMP and 1-MeAde. Basal levels of intracellular cGMP were only about 20% of those of cAMP and were not influenced by treatment with GSS. At 1 mM, 3-isobutyl-1-methylxanthine (IBMX), a potent phosphodiesterase inhibitor, stimulated both 1-MeAde and cAMP production in a concentration-dependent manner. Con A and two serine proteases also raised both cAMP and 1-MeAde production. Con A-induced (1.0 mg/ml) increases in cAMP and 1-MeAde were greater than the response to GSS (0.1 mg/ml) and were completely suppressed by treatment with alpha-methyl-D-mannoside (10 mM), a competitive inhibitor of Con A. These results strongly suggest that cAMP is a second messenger in the production of 1-MeAde by starfish ovarian follicle cells.

Fatty chain composition of phospholipids in sea urchin spermatozoa

1. An analysis was made of lipids extracted from the spermatozoa of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina. 2. Nearly all the lipids from both species consisted of phospholipids (about 80%) and cholesterol (about 14%). Triglyceride and cholesterol ester were present in trace amounts. 3. The fatty acid composition of each phospholipid was determined by gas-liquid chromatography. In both species, the fatty acid consisting of phosphatidylethanolamine was of the unsaturated type for the most part, while cardiolipin was comprised to a considerable degree of saturated fatty acids. In phosphatidylcholine and phosphatidylserine from H. pulcherrimus sperm, unsaturated fatty acid content was somewhat higher than that in phospholipids from A. crassispina sperm.

Inhibitory Effect of a SALMFamide Neuropeptide on Secretion of Gonad-Stimulating Substance from Radial Nerves in the Starfish Asterina pectinifera

Abstract In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.

Phosphatidylcholine metabolism for energy production in sea urchin spermatozoa.

Sea urchin spermatozoa use endogenous phosphatidylcholine (PC) to produce energy for swimming. The catabolism of PC was studied in Hemicentrotus pulcherrimus spermatozoa. Following incubation in sea water, the content of PC decreased and that of choline increased gradually, whereas phosphocholine maintained a constant level. Measurement of the radioactivity in metabolites converted from 1-palmitoyl-2-[1-14C]linoleoyl-PC, [choline-methyl-14C]dipalmitoyl-PC and 1-[1-14C]palmitoyl-lysophosphatidylcholine (LysoPC) showed that the major degradative pathway is PC----LysoPC----glycerophosphocholine----choline. 1-Palmitoyl-2-[1-14C]linoleoyl-PC and [1-14C]oleic acid were oxidized to 14CO2 in a cell-free system of spermatozoa. Sea urchin spermatozoa thus appear to quite likely obtain energy through the oxidation of fatty acid(s) from PC.

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.

In Vitro Induction of Starfish Oocyte Maturation by Cysteine Alkylesters

The effect of various disulfide‐reducing agents including cysteine and its alkylesters on the induction of germinal vesicle breakdown (GVBD) in starfish (Asterina pectinifera) oocytes was investigated in vitro. Although cysteine did not induce GVBD, its alkylesters were effective. Cysteine alkylesters significantly mimicked the effect of 1‐methyladenine (1‐MeAde), the naturally occurring maturation‐inducing hormone of starfish, on oocyte maturation. However, the effective concentrations and pH optimum for stimulation of oocyte maturation varied between 1‐MeAde and the cysteine alkylesters. By comparing pKa values of the disulfide‐reducing agents to pH of the medium, it is suggested that the redox potential of a disulfide‐reducing agent is an important indicator its ability to induce oocyte maturation.

Degeneration of Respiratory System in Sea Urchin Spermatozoa during Incubation in Seawater for Long Duration

Abstract Motility and respiration were examined in spermatozoa of the sea urchin Hemicentrotus pulcherrimus after dilution and incubation in seawater at pH 8.2 at 20°C. Almost all spermatozoa were motile during 12 hr of incubation, but their respiratory rate decreased gradually. The acrosome reaction was also induced by egg jelly solution during 12 hr of incubation in seawater. However, the ratio of spontaneous acrosome reacted spermatozoa was quite low during the same period. An intracellular pH (pHi) of spermatozoa was about 7.5 just after dilution in seawater and was almost constant during 12 hr of incubation. Upon dilution and incubation in seawater, activity of NADH-cytochrome c reductase decreased in propotion to the decrease in the respiration in spermatozoa, whereas cytochrome c oxidase activity was hardly changed. These suggest that the degeneration of respiratory system during 12 hr of incubation in seawater is due to the decrease in the NADH-cytochrome c reductase activity. In energy production system, phosphatidylcholine as a preferred substrate was efficiently hydrolyzed during 4 hr of incubation and then the activity of the energy metabolism decreased gradually. Beyond 12 hr incubation in seawater, the number of immotile spermatozoa increased and respiratory rate declined rapidly. Also, the percentage of the acrosome reaction induced by the egg jelly solution decreased. These are probably due to the increase in the number of dead spermatozoa after 12 hr of incubation in seawater. It is thus concluded that the life-span of H. pulcherrimus spermatozoa is about 12 hr after dilution in seawater.

Methylation during 1-methyladenine production by starfish ovarian follicle cells

Abstract 1. 1. Under the influence of gonad-stimulating substance (GSS), ovarian follicle cells of the starfish, Asterina pectinifera , produce 1-methyladenine (1-MeAde) for resumption of meiosis in oocytes. As a methyl donor, methionine increased GSS-induced 1-MeAde production in follicle cells, but 5-methyltetrahydrofolate or homarine had no effect. 2. 2. 1-Methyladenine production by GSS in follicle cells was enhanced by l -methionine, d -methionine and seleno- dl -methionine. Without GSS, however, these chemicals alone did not induce 1-MeAde production. 3. 3. Both l - and d -ethionine, competitive inhibitors of methionine, reduced 1-MeAde levels produced by follicle cells in the presence of GSS. Seleno- dl -ethionine also inhibited 1-MeAde production. l -Methionine, d -methionine and seleno- dl -methionine abolished the ethionine-induced inhibition of 1-MeAde production.