Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*: (starfish oocyte maturation/tumor promoters/phorbol esters/teleocidin/retinoids).

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml.

Regulation of Sperm Motility in Starfish. I. Initiation of Movement: (starfish sperm/seminal plasma/sperm propulsion/sperm best frequency).

Starfish seminal plasma has such characteristics as higher concentration of potassium ions, higher osmolality, and lower pH compared with those of sea water. These factors independently inhibited the movement of spermatozoa. Sperm movement was recorded by taking photographs of the swimming paths under a dark field microscope.

Oocyte Maturation in Starfish: Sequential Changes of Oocyte Shape Induced by 1-Methyladenine Associated with Germinal Vesicle Breakdown: (oocyte maturation/starfish/1-methyladenine).

Observation of the early events of starfish oocyte maturation revealed a sequential change of the oocyte shape induced by 1-methyladenine (1-MeAde). The lengths of two diameters of the whole oocyte, one parallel to the egg axis (a-v diameter) and the other which is perpendicular to a-v diameter (e diameter) were measured by taking photographs successively. About 10 min after 1-MeAde administration, a sudden decrease of the a-v diameter (shortening of oocyte) occurred followed by a sudden increase (elongation of oocyte).

Cytoplasmic Maturity Revealed by the Structural Changes in Incorporated Spermatozoon during the Course of Starfish Oocyte Maturation.

Immature oocytes of the starfish, Asterina pectinifera, are polyspermic. Spermatozoa can enter immature oocytes upon insemination, but the changes associated with the fertilization process in oocytes matured with 1-methyladenine (1-MeAde), such as the formation of aster and pronucleus, were not observed. After immature oocytes, previously inseminated, were matured with 1-MeAde, the formation of the sperm monaster was observed during germinal vesicle breakdown (GVBD).

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.

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.

EFFECT OF LOCAL APPLICATION OF 1-METHYLADENINE ON THE SITE OF POLAR BODY FORMATION IN STARFISH OOCYTE.

Mechanism by which the site of polar body formation is determined in starfish oocytes was investigated in relation to the action of 1-methyladenine (1-MeAde). Local staining with Nile Blue of Asterina pectinifera oocytes revealed that there exists a prospective site of polar body formation (PSPBF) on the nearest surface to the position of germinal vesicle. The site of polar body formation was found to shift to some extent from PSPBF toward the area locally applied with 1-MeAde, suggesting that the actual site of polar body formation is not determined yet at the germinal vesicle stage.

INDUCTION OF OOCYTE MATURATION BY DISULFIDE-REDUCING AGENT IN THE SEA CUCUMBER, STICHOPUS JAPONICUS.

1-Methyladenine (1-MeAde) is known to be a natural inducer of starfish oocyte maturation. Disulfide-reducing agents such as dithiothreitol (DTT) and 2, 3-dimercapto-1-propanol (BAL) are known to mimic the action of 1-MeAde in inducing starfish oocyte maturation. Although 1-MeAde failed to induce oocyte maturation in sea cucumbers, breakdown of germinal vesicles and subsequent meiotic behaviour of chromosomes were induced by the treatment with DTT in the pronase-treated oocytes of the sea cucumber, Stichopus japonicus.

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.

BINDING OF CONCANAVALIN A TO THE SURFACE OF STARFISH FOLLICLE CELLS AND PRODUCTION OF 1-METHYLADENINE.

Starfish follicle cells, treated with concanavalin A (Con A), continued to produce 1-methyl-adenine (1-MeAde), an inducer of starfish oocyte maturation, after rinsing with artificial seawater (ASW). On the other hand, they ceased to produce the substance if treated with methyl α-Dmannoside (αMM). These cells produced again 1-MeAde when re-stimulated with Con A after removal of αMM.

DISTRIBUTION OF MATURATION-PROMOTING FACTOR IN STARFISH OOCYTE STRATIFIED BY CENTRIFUGATION.

In starfish, cytoplasm taken from maturing oocytes is capable of inducing breakdown of the germinal vesicle and subsequent maturation when injected into immature oocytes. The cytoplasmic factor has been designated as "maturation-promoting factor" (MPF). Ooplasm was stratified by centrifugation of maturing oocytes in density-graded Ficoll-seawater, without disrupting the cell.

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.

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.

FORMATION OF THE FERTILIZATION MEMBRANE BY INSEMINATION OF IMMATURE STARFISH OOCYTES PRETREATED WITH CALCIUM-FREE SEAWATER.

When immature oocytes of the starfish, Asterina pectinifera, were treated with calcium-free seawater for 1 hr and then inseminated in normal seawater, they formed several blisters, indicative of polyspermy, and raised fertilization membranes. These oocytes continued to have intact germinal vesicles. Electron microscopic study revealed that the egg surface remained virtually unchanged after the treatment with calcium-free seawater.

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.

INDUCTION OF SPAWNING AND OOCYTE MATURATION IN STARFISH BY 1-METHYL-ADENOSINE MONOPHOSPHATE.

l-Methyladenosine monophosphate (l-McAMP) induces ovulation and oocyte maturation when applied to isolated ovarian fragments of Asterina pectinifera. However, isolated oocytes fail to mature even in the presence of this substance. When ovarian or testis fragments are incubated with l-McAMP, the supernatant of the incubation mixture acquires the maturation-inducing activity.