Influence Of Gonad-stimulating Substance Research Articles

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-(14)C]methionine (Met), there was an increase in the level of radiolabeled S-adenosylmethionine (SAM). After further incubation with GSS, the [methyl-(14)C]SAM level decreased, concomitant with a marked increase in radiolabeled 1-MeAde production. The amount of [methyl-(14)C]SAM consumed under the influence of GSS was similar to the amount of [methyl-(14)C]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.

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.

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.

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.

1-Methyladenine production by ovarian follicle cells responsible for spawning in the starfishAsterina pectinifera

Summary Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of gonad-stimulating substance (GSS). It has also been reported that 1-MeAde induces follicular envelope breakdown (FEBD), releasing oocytes from the ovaries; i.e., spawning. This study was undertaken to determine whether the amount of 1-MeAde produced by follicle cells is enough to induce germinal vesicle breakdown (GVBD) and FEBD in the starfish Asterina pectinifera. After incubation of an ovary with GSS at concentrations of more than 0.05 mg/ml for about 20 min, spawning started. Almost the same period of time was required for spawning after treatment with 0.1 μM 1-MeAde. However, continuous treatment with GSS was not necessary, and about 5 min of GSS application was sufficient for induction of spawning. Each oocyte is surrounded by a follicular envelope consisting of about 50 cells. 1-MeAde production by isolated follicle cells started immediately upon GSS st...

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.

Methylation during 1-methyladenine production by starfish ovarian follicle cells

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.

1-Methyladenine-producing cell in starfish testis

The site of production of 1-methyladenine (1-MeAde) in the testis of the starfish, Asterina pectinifera, was investigated. Incubation of spermatozoa with the gonad-stimulating substance (GSS) revealed that spermatozoa do not have any 1-MeAde-producing activity. After removing most of the spermatozoa, the testis components were divided into testicular wall fraction and free cell fraction. When their 1-MeAde-producing activity was compared, the free cell fraction showed higher activity than the wall fraction. The free cell fraction contained mainly two types of cells: A cells, 7–10 μm in diameter and with granular cytoplasm, and B cells, 4–5 μm and with clear cytoplasm. The purified A cell fraction was found to produce 1-MeAde under the influence of GSS, whereas the B cell fraction failed to produce the substance. It is therefore concluded that the site of production of 1-MeAde in starfish testes is the A cell. Electron microscopic observation of A-cells revealed that these 1-MeAde-producing cells are non-germinal and phagocytotic, and are located along the germinal epithelium of the testis. They are also found among the developing germ cells and spermatozoa in the center of the testis.

Neosynthesis of 1-methyladenine in the starfish gonad under the influence of gonad-stimulating hormone

When homogenate of starfish ovary is hydrolysed with 0.6 N hydrochloric acid for 30 min at 100 °C, maturation-inducing substance, 1-methyladenine, is liberated in the hydrolysate. This is confirmed by thin layer chromatography. Without hydrolysis, the ovarian homogenate does not show any maturation-inducing activity. When the soluble ribonucleic acid (sRNA) obtained from the ovary of Asterias amurensis is hydrolysed under similar conditions, the hydrolysate induces oocyte maturation, and thin layer chromatography shows that it contains 1-methyladenine, suggesting that 1-methyladenine is liberated from sRNA. Without hydrolysis, sRNA has no maturation-inducing characteristic. The gonad-stimulating substance (GSS), responsible for the production of 1-methyladenine in starfish gonads under physiological conditions, has no effect on the activity of ovarian ribonuclease which acts on the sRNA, suggesting that 1-methyladenine produced in the presence of this hormone does not come from sRNA which preexists in the ovary. The amount of 1-methyladenine liberated in the hydrolysate of the mixture of ovary and GSS, preincubated before hydrolysis, corresponds to the sum of those of ovarian hydrolysate and the incubation mixture of ovary and GSS without hydrolysis. These data suggest that 1-methyladenine produced under the influence of GSS is not a breakdown product of some 1-methyladenine-containing substance such as sRNA present in the ovary, but is newly synthesized.

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% (10 4 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 (10 4/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.