METHIONINE-ACTIVATING ENZYME IN STARFISH FOLLICLE CELLS *

In starfish follicle cells 1-methyladenine is produced under the influence of a gonad-stimulating hormonal peptide (GSS). Since such production of the substance is enhanced by the addition of L-methionine or S-adenosylmethionine in vitro, the presence of methionine-activating enzyme in the follicle cells of the starfish, Asterina pectinifera, was investigated. To detect enzyme activity, the enzyme was partially purified from the supernatant of the follicle-cell homogenate by precipitation with ammonium sulfate followed by gel-filtration on a Sephadex G-150 column. Using such a preparation of the enzyme, the production of S-adenosylmethionine from L-methionine and adenosine triphosphate was clearly demonstrated by thin-layer chromatography. GSS was found to exert no effect on the activity of the methionine-activating enzyme. The hormonal peptide, GSS, is therefore considered to take part in some reaction other than this step in the formation of 1-methyladenine.     

INHIBITION OF GERMINAL VESICLE BREAKDOWN AND POLAR BODY FORMATION BY NICOTINAMIDE IN STARFISH AND SURF CLAM OOCYTES*

Nicotinamide inhibited both germinal vesicle breakdown (GVBD) and polar body formation (PBF) in surf clam and starfish oocytes. In the surf clam nicotinamide at 0.3 mM completely blocked PBF in the fertilized oocytes. For blockage of GVBD higher concentration was required. In the starfish, nicotinamide (30 mM) prevented PBF but not GVBD, when added 7 min after the commencement of 1-methyladenine (1-MeAde) administration. These results suggest that PBF is blocked by nicotinamide independent of its effect on GVBD. In the case of starfish, NAD⁺was more effective than nicotinamide in inhibiting oocyte maturation. Nicotinamide also blocked GVBD induced by microinjection of the cytoplasm containing maturation-promoting factor (MPF) obtained from 1-MeAde-treatcd oocytes. These results suggest that nicotinamide prevents the action of MPF rather than inhibiting the interaction of 1-McAde with cell membrane or the induction of MPF.     

Genetic and Biochemical Studies on Ommochrome Genesis in an Albino Strain of a Terrestrial Isopod,Armadillidium vulgare

Genetic studies and quantitative determination of levels of 3-hydroxykynurenine and kynurenine were performed in an albino strain of a terrestrial isopod Armadillidium vulgare. From the results of matings between the albino and the albino, the red, the dark red, or the wild type individuals, the albino A. vulgare seems to be regulated by an autosomal gene(s) recessive to its wild allele. Litter mating of F₁ progenies obtained by crossing the albino and the red mutant or the albino and the dark red mutant yielded progenies at a ratio of 3:6:3:4 for the red, the dark red, the wild, and the albino phenotypes, respectively. The albino gene(s) seems not to be allelic but to be epistatic to the red gene(s) with respect to ommochrome biosynthesis. Quantitative determination of 3-hydroxykynurenine carried out by high-performance liquid chromatography with electrochemical detection revealed that the 3-hydroxykynurenine content in the albino was significantly lower than that in the wild or the red type. The whole content of 3-hydroxykynurenine after enzymatic conversion of kynurenine to 3-hydroxykynurenine was still considerably lower than that found in the wild type, even though it increased after the conversion. The albino gene(s) seems to be associated with a blockage at distinct level(s) of ommochrome biosynthesis.     

Concanavalin A-Induced Morphological Changes and Its Binding Sites in Starfish Follicle Cells as Revealed by Electron Microscopy

Concanavalin A (Con A) stimulates the production in starfish follicle cells of 1-methyladenine, a hormone which induces oocyte maturation. We have therefore investigated Con A-induced morphological changes and Con A-binding sites in the follicle cell using native Con A and horseradish peroxidase- or ferritin-labeled Con A (HRP-Con A, Fer-Con A). After isolated follicle cells were incubated with Con A (1 mg/ml), vacuoles, the Golgi complex and multivesicular body-like organelles (MVBs) became prominent in most of the cells. After follicle cells were prefixed and then incubated with Fer-Con A for 60 min, tagged ferritin was diffusely and randomly distributed as single or small clustered particles on the cell surface. The incubation of isolated follicle cells with Fer-Con A for 10 min before fixation resulted in numerous ferritin particles localized along the internalized membrane, and also in vacuoles, MVBs and small lysosome-like structures. After 60 min incubation with Fer-Con A, ferritin was further located in large lysosome-like structures and in vesicles near and in the Golgi area as well as in the organelles described above. HRP-Con A binding sites were also observed in vacuoles and MVBs of the intact cells.
These results suggest that Con A binds at first to the cell surface and causes rapid internalization and that membrane-bound Con A is easily endocytosed into vacuoles, MVBs and lysosome-like structures, and is later incorporated in some vesicles in the Golgi area.     

MECHANISM OF STARFISH SPAWNING. III. PROPERTIES AND ACTION OF MEIOSIS-INDUCING SUBSTANCE PRODUCED IN GONAD UNDER INFLUENCE OF GONAD-STIMULATING SUBSTANCE

The gonad-stimulating substance (GSS) contained in nerve extract of the starfish, Asterina pectinifera, acts on the ovary to produce an active substance responsible for oocyte maturation, meiosis-inducing substance (MIS). MIS was successfully separated from GSS by gel-filtration on Sephadex. The MIS fraction had also spawning-inducing activity. Starfish testis also produced MIS under the influence of GSS. MIS production was shown in six starfish species. Although some species specificity characterizes GSS, this was not observed in MIS. Production of MIS in the ovary was found to begin immediately after the addition of GSS. The amount of MIS produced increased as the concentration of GSS was raised, and the longer the time of treatment with GSS, the greater was the amount of MIS produced. MIS was rather heatstable, insoluble in ether, benzene and petroleum ether but soluble in 95% ethanol. Its activity was not destroyed by pronase. Injection of MIS into the coelomic cavity induced gamete-shedding in both male and female. However, MIS failed to induce spawning when applied from the outside of the body. Both GSS and MIS were found in the coelomic fluid only when the starfish were undergoing natural spawning. A mechanism of starfish spawning, based on the action of GSS and MIS is discussed.     

PRESENCE OF 1-METHYLADENINE IN SEA URCHIN GONAD AND ITS RELATION TO OOCYTE MATURATION

A water extract of sea urchin ovary was found to induce maturation of starfish oocytes in vitro . The presence of the active substance was demonstrated in ovaries of the sea urchins, Pseudocentrotus depressus, Anthocidaris crassispina and Hemicentrotus pulcherrimus , and the sand dollars, Clypeaster japonicus and Peronella japonica . The active substance was also contained in the testes of these echinoids. That the content of this substance increases during the reproductive season was demonstrated with Anthocidaris gonads. The active substance present in ovary or testis of the sea urchins was successfully extracted with 85% ethanol and purified with gel-filtrations on Sephadex G-15 columns after washing with chloroform and ether. The purified active substances were the same and were identified as 1-1-methyladenine by thin layer chromatography. 1-Methyladenine was found to be effective in inducing oocyte maturation in Anthocidaris crassispina in vitro . Therefore, 1-methyladenine seems to play an important role in oocyte maturation in echinoids as well as in asteroids.     

Effect of 17β-Estradiol on Growth of Oocytes in Cultured Ovarian Fragments of the Starfish, Asterina Pectinifera

The development of growing oocytes of the starfish, Asterina pectinifera , was divided into five stages according to their histological features. Specimens collected monthly from Rishiri Island throughout a year were used. The effect of 17 β-estradiol on oocyte growth was investigated. When ovarian fragments taken from April females were cultured in artificial seawater containing Medium 199 and streptomycin sulfate for 3 days, oocytes within such fragments did not show any changes in diameter. However, when similar ovarian fragments were cultured in the presence of 10⁻⁵M 17 β-estradiol, the oocyte diameters increased significantly. It is concluded that 17 β-estradiol enhances the growth of starfish oocytes in cultured ovarian pieces.