EFFECTS OF METHIONINE AND S-ADENOSYLMETHIONINE ON PRODUCTION OF 1-METHYLADENINE IN STARFISH FOLLICLE CELLS

Incubation of follicle cells of the starfish ( Asterias amurensis ) with a gonad-stimulating hormonal peptide (GSS) leads to the production of 1-methyladenine. the trigger of oocyte maturation. Addition of L-methionine to the incubation medium promotes the production of 1-methyladenine. This study shows that S -adenosylmethionine also enhances the production of 1-methyladenine in such an incubation mixture. However, in the absence of GSS, addition of S -adenosylmethionine failed to produce an appreciable amount of 1-methyladenine. When an homogenate of isolated follicle cells was incubated, a certain amount of 1-methyladenine was produced, whether or not GSS was present. Addition of S -adenosylmethionine to the incubation mixture of follicle homogenate enhanced the production of 1-methyladenine. Although addition of adenosine triphosphate (ATP) to such an incubation mixture had little effect in producing 1-methyladenine, it exerted a promoting effect on 1-methyladenine production when S -adenosyl-methionine was present. These results suggest that methionine, through its active form, S -adenosylmethionine, acts as a donor of methyl group in the formation of 1-methyladenine.     

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

Summary

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). With respect to 1-MeAde production by follicle cells of the starfish, Asterina pectinifera, (1) the action of GSS is initiated by a receptor mediated activation of G-proteins, resulting in the activation of adenylate cyclase and cyclic AMP (cAMP) formation; (2) 1-MeAde produced under the influence of GSS is not prestored within the follicle cells but is newly synthesized from a 1-MeAde precursor; (3) AMP plays an important role in the process of methylation during 1-MeAde biosynthesis induced by GSS.     

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.     

PRODUCTION OF 1-METHYLADENINE INDUCED BY CONCANAVALIN A IN STARFISH FOLLICLE CELLS

Concanavalin A (Con A) was found to induce maturation of oocytes with follicular envelopes in the starfish, Asterina pectinifera . Treating a Con A sample with 85% ethanol and heat revealed that the maturation-inducing activity of the sample was not due to possible contamination with 1-methyladenine, but to Con A itself. However, Con A had little maturation inducing effect on isolated oocytes from which the follicular envelope had been removed, suggesting that its effect is indirect and probably mediated by the follicle cells. When follicle cells were incubated in seawater containing Con A, a maturation-inducing substance was found to have been produced in the incubation medium. This was purified and identified as 1-methyladenine. Therefore it is concluded that Con A has the same capacity as GSS, a gonad-stimulating peptide hormone of neural origin, to induce production of the maturation-inducing substance. Other plant lectins such as phytohemagglutinin P and wheat germ agglutinin had little effect in inducing production of 1-methyladenine in follicle cells.     

Maturation-Inducing Substances in Asteroid and Echinoid Oocytes

In starfish a hormonal peptide, gonad-stimulating substance(GSS), which is released from nervous tissue, acts on the gonadto produce a maturation-inducing substance (MIS), an inducerof oocyte maturation and spawning. MIS is 1-methyladenine (1-MA).This substance acts on the surface of the oocyte. Cytoplasmicmaturation as revealed by fertilizability is also induced by1-MA. The amount of 1-MA can be determined very accurately witha bioassay method using isolated oocytes. In 1-MA formation,GSS seems to enhance the methylation of some compound whichcontains a purine nucleus at its N1 site. The methyl donor isprobably S-adenosylmethionine. 1-Methylated precursor seemsto be transformed to 1-methyl AMP and then hydrolyzed into 1-methyladenosineand phosphate by phosphomonoesterase. 1-Methyladenosine is finallysplit into 1-MA and ribose by 1-methyladenosine ribohydrolase.So-called spontaneous maturation of oocytes isolated in seawater is due to the action of 1-MA produced in follicle cellseven in the absence of GSS. 1-MA is present in echinoid gonadsand seems to act as MIS also in these animals. Disulfidereducingagents such as dithiothreitol and 2,3-dimercapto-1-propanolinduce starfish oocyte maturation. On the other hand, sulfhydrylreagents such as p-chloromercuribenzoate, iodoacetamide, andN-ethylmaleimide suppressed 1-MA-induced oocyte maturation.Since Concanavalin A acts on the follicle cells to produce 1-MA,the action of this substance seems to be quite similar to thatof GSS.     

Change in the levels of adenine-related compounds in starfish ovarian follicle cells following treatment with gonad-stimulating substance

The resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeA), which is produced by ovarian foilicle cells under the influence of a gonad-stimulating substance (GSS). It has been reported that the 1-MeA produced is newly synthesized via a process of methylation, rather than being pre-stored within follicle cells or a breakdown product of some 1-MeA-containing substance. The present study examined a possible substrate for 1-MeA biosynthesis stored in follicle cells of the starfish Asterina pectinifera . Analyses using high-performance liquid chromatography indicated a large source of ATP among the adenine-related compounds in these follicle cells. When follicle cells were incubated in seawater in the presence of GSS, 1-MeA production was stimulated significantly. GSS also caused a reduction in intracellular levels of ATP. There was no change in the levels of either ADP or AMP. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeA produced. Methionine and selenomethionine enhanced both 1-MeA production and ATP consumption by GSS in follicle cells. In contrast, ethionine and selenoethionine, competitive inhibitors of methionine, inhibited these processes. These results suggest that ATP is a possible substrate in the biosynthesis of 1-MeA by starfish ovarian follicle cells.     

Methyl donor for 1‐methyladenine biosynthesis in starfish ovarian follicle cells

1‐Methyladenine (1‐MeAde), the oocyte maturation‐inducing substance of starfish, is produced by ovarian follicle cells upon stimulation with a gonad‐stimulating substance (GSS) released from radial nerves. It has been reported that a process of methylation is involved in GSS‐induced 1‐MeAde production by starfish ovarian follicle cells. The present study sought to identify a possible methyl donor for 1‐MeAde biosynthesis in follicle cells of the starfish Asterina pectinifera. When isolated follicle cells were incubated with [methyl‐¹⁴C]methionine (Met), there was an increase in the level of radiolabeled S‐adenosylmethionine (SAM). After further incubation with GSS, the [methyl‐¹⁴C]SAM level decreased, concomitant with a marked increase in radiolabeled 1‐MeAde production. The amount of [methyl‐¹⁴C]SAM consumed under the influence of GSS was similar to the amount of [methyl‐¹⁴C]1‐MeAde produced. Therefore, it is concluded that SAM is a methyl donor for 1‐MeAde biosynthesis in starfish ovarian follicle cells. On the other hand, it is likely that the purine molecule of 1‐MeAde is not derived from SAM but from ATP. 3‐Isobutyl‐1‐methylxanthine, a potent inhibitor of cyclic AMP phosphodiesterase, also caused a reduction in the level of radiolabeled SAM following 1‐MeAde production. This suggests that the methylation process of 1‐MeAde biosynthesis in starfish ovarian follicle cells upon stimulation with GSS is mediated by a second messenger, cyclic AMP. Mol. Reprod. Dev. 54:63–68, 1999. © 1999 Wiley‐Liss, Inc.     

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera

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.     

Effect of Ca2+-free Seawater Treatment on 1-Methyladenine Production in Starfish Ovarian Follicle Cells

When follicle cells from ovaries of the starfish Asterina pectinifera were washed with Ca²⁺-free seawater (CaFSW), the production of 1-methyladenine (1-MeAde) in response to gonad-stimulating substance (GSS) decreased to a large degree. The cyclic AMP content of CaFSW-treated follicle cells was much lower than that of non-treated cells, and the level was increased slightly by GSS, but not to a degree sufficient for production of 1-MeAde. The production of 1-MeAde and cyclic AMP in the presence of GSS was dependent on extracellular Ca²⁺ concentration, and was considerably reduced at a Ca²⁺ concentration below 1 mM. Thus, the decrease of 1-MeAde production by follicle cells after treatment with CaFSW is due to the low levels of cAMP. Furthermore, an ADP-ribosylation experiment using [α-³²P]NAD⁺ in the presence of cholera toxin and pertussis toxin with membrane preparations of follicle cells treated with CaFSW revealed the presence of two types (stimulatory and inhibitory) of G-proteins. However, activity of the adenylyl cyclase was not influenced by GSS regardless of the presence or absence of GTP. These findings may suggest that GSS is unable to bind to its receptor in follicle cells after washing with CaFSW.     

Absence of 1-Methyladenine Production in Follicle Cells Obtained from Starfish Ovaries in the Post-Spawning Season

Follicle cells from the ovaries of starfish Asterina pectinifera collected in the breeding season and then kept in an aquarium for three months did not produce 1-methyladenine (1-MeAde) in response to genad-stimulating substance (GSS). The cyclic AMP content of follicle cells of the ovaries was much lower in the post-spawning season than in the spawning season. In the post-spawning season, the cyclic AMP level was increased slightly by GSS, but nor insufficiently for production of 1-MeAde. Addition of 3-isobutyl-1-methylxanthine, a potent phosphodiesterase inhibitor, stimulated the productions of GSS-induced 1-MeAde and cyclic AMP. Adenylate cyclase in membrane preparations of follicle cells from ovaries in the post-spawning season was stimulated by nonhydrolyzable GTP analogs and forskolin. An experiment on ADP-ribosylation with [α-³²P]NAD in the presence of cholera toxin and pertussis toxin showed the presence of two types (stimulatory and inhibitory) of guanine nucleotide-binding regulatory proteins (G-proteins). However, GSS has no effect on the adenylate cyclase activity regardless of the presence of GTP. These findings suggest that GSS does not bind to its receptor in follicle cells of the overy in the post-spawning season, although these cells possess G-proteins and adenylate cyclase. Thus the absence of 1-MeAde production by follicle cells obained from ovaries in the post-spawning season appears to be due to lack of receptor protein for GSS.     

Characterization and action of meiotic maturation inhibitors in starfish ovary

Mechanical release of oocytes from the ovary of the starfish Asterias amurensis into sea water results in “spontaneous” meiotic maturation of the oocytes. The substances blocking the maturation of Asterias oocytes have been purified from the ovary and shown to be steroid glycosides named asterosaponins A and B. The extract prepared from isolated oocytes was incapable of inhibiting oocyte maturation. The ovarian extract inhibited the production of 1-methyladenine (1-MA) in follicle cells surrounding the oocyte. The ovarian extract failed to influence 1-MA-induced maturation of the oocyte with or without follicle cells. It can be concluded from the present results that the role of the ovarian extract containing steroid glycosides is to arrest “spontaneous” production of 1-MA in follicle cells. The suppression can be overcome by the action of a gonadotropic peptide hormone released from the nerve tissue.     

Effect of Cysteine and Its Derivatives on 1-Methyladenine Production by Starfish Follicle Cells

Spawning of the gametes in the starfish, Asterina pectinifera , induced by a gonad-stimulating substance (GSS) was inhibited by cysteine. This inhibitory effect of cysteine was due to the reduction of GSS activity and the inhibition of 1-methyadenine (1-MeAde) production in follicle cells. Homocysteine and cysteamine also reduced GSS activity, but cystamine had no effect on the activity. This suggests that the loss of GSS activity is related to a SH group. Furthermore, the effect of cysteine on 1-MeAde production was investigated using isolated follicle cells. The half maximum decrease of the GSS-dependent 1-MeAde production in follicle cells was obtained with the concentration of 2.0 mM L-cysteine. A similar effect was observed with D-cysteine. Homo cysteine and cysteamine also reduced the GSS-dependent 1-MeAde production. But methionine, serine, glutamic acid and aspartic acid did not affect the 1-MeAde production of follicle cells. In addition, cystamine also inhibited the GSS-dependent 1-MeAde production. These suggest that both a SH group and a SS bond of these compounds are effective on the inhibition of the GSS-dependent 1-MeAde biosynthesis. Cysteine and homocysteine also inhibited concanavalin A (Con A).induced 1-MeAde production of follicle cells, but they had no effect on binding of Con A to the surface of follicle cells.     

1-Methyladenine production from ATP by starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance in starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves. We have shown previously that GSS causes a reduction in the intracellular levels of ATP coincident with 1-MeAde production. The present study examined whether the adenine molecule of 1-MeAde is directly derived from ATP. When isolated follicle cells from the starfish Asterina pectinifera were preloaded with [U-14C]adenine or [U-14C]adenosine, there was an increase in the intracellular levels of radiolabeled adenine nucleotides, particularly ATP. Following further incubation with GSS, the intracellular levels of radiolabeled ATP decreased, concomitant with a marked increase in the levels of [14C]1-MeAde in the medium. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeAde produced. However, there was no change in the levels of ADP and AMP regardless of the presence or absence of GSS. These findings strongly suggest that 1-MeAde is synthesized from ATP as a substrate in follicle cells under the influence of GSS. Furthermore, using [methyl-3H]methionine, the methyl group of 1-MeAde was found to be derived from methionine. Thus GSS appears to stimulate the synthesis of 1-MeAde from ATP via the methylation process in starfish ovarian follicle cells.     

Site of production of meiosis-inducing substance in ovary of starfish

The site of production of meiosis-inducing substance (MIS), produced in the ovary under the influence of gonad-stimulating substance (GSS) taken from radial nerves, was studied with the starfish, Asterina pectinifera. The rate of oocyte maturation observed in isolated oocytes with follicles kept in sea water containing GSS (100 μg/ml) was generally low when a small number of eggs per unit quantity of sea water was used. However, with increased number of oocytes per milliliter of GSS-sea water, the maturation rate increased up to 100% (10⁴ eggs/ml). The supernatant of such an incubation mixture of oocytes and GSS contained an appreciable amount of MIS. When a large number of oocytes (10⁴/ml) was used, the rate of oocyte maturation increased with the rise in concentration of GSS. Isolated follicles were found to produce MIS when incubated in sea water containing GSS, suggesting that the site of production of MIS is the follicle cells. The physiological role of the follicle cells surrounding the full grown oocytes seems to be to produce MIS just before spawning under the influence of GSS.     

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.     

BIOSYNTHESIS OF S-ADENOSYL-L-METHIONINE IN THE RAT PINEAL GLAND

This investigation provides direct evidence that rat pineal gland contains ATP:L-methionine S-adenosyltransferase. The specific activity of the enzyme is about 25 times greater than that of hydroxyindole-O-methyltransferase, the unique marker enzyme in this tissue. The major product of the reaction catalyzed by ATP:L-methionine S-adenosyltransferase has been identified as S-adenosyl-methionine which can function as a methyl group donor for the formation of melatonin. The specific activity of pineal ATP:L-methionine S-adenosyltransferase is at least eight times greater than that of brain, and one-half that of the liver enzyme.     

TRANS-SULPHURATION IN PRIMATE BRAIN: REGIONAL DISTRIBUTION OF METHIONINE-ACTIVATING ENZYME IN THE BRAIN OF THE RHESUS MONKEY AT VARIOUS STAGES OF DEVELOPMENT

—The regional distribution of methionine-activating enzyme (ATP:l-methionine S-adenosyltransferase; EC 2.4.2.13) in the brain of the Rhesus monkey was determined at various stages of development. Activity of the methionine-activating enzyme was highest in pituitary gland, cerebellum and occipital grey matter, and lowest in areas rich in white matter: spinal cord, subcortical white matter, corpus callosum and optic chiasm. There was no marked change in activity in any area during development from the first-trimester foetus to the juvenile animal. During the same period of development, activity of the methionine-activating enzyme in the liver increased approximately four-fold. The findings are discussed in relation to those transmethylating enzymes and/or methylated products which have been studied in mammalian brain. The presence of high activity of the methionine-activating enzyme in cerebellum and occipital grey matter suggests that previously unrecognized methylating processes may be important in the metabolism of these areas of brain.     

Site of action of 1-methyladenine in inducing oocyte maturation in starfish : Kinetic evidence for receptors localized on the cell membrane

The cellular localization of the receptors for 1-methyladenine, the natural substance inducing nuclear maturation in starfish oocytes, has been investigated in Marthasterias glacialis, by comparing its biological activity when used at limiting concentration, either in presence or in absence of specific inhibitors of its uptake. Absence of difference in the biological effect under these conditions argues strongly for an external localization of the hormonal receptors.     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

Oocyte-follicle cell relationships in a starfish

Summary The follicle cells which surround the oocytes of starfish are known to both release the hormone 1-methyladenine and to respond to it by an active movement which forms a component of the spawning response to the hormone. In Patiria miniata these flagellated cells are located peripheral to the oocyte and have long cytoplasmic processes which penetrate the vitelline layer to the egg surface to form an adhering zonule-like junction. Within the follicle cell cytoplasm are located elongate filamentous bands which appear to represent a component of the contractile mechanism that mediates follicle cell response to 1-methyladenine. These bands do not resemble the filaments of vertebrate smooth muscle cells (quantity, distribution and size of filaments; lack of dense bodies in the filament mass), nor the contractile units of the superficial epithelium of lower vertebrate follicles.This investigation was supported by grants HD-07194 and HD-12499 from the National Institutes of Health. We are indebted to Mr. James D. Huber for able technical assistance