ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Significance of an Increase of Intracellular Adenosine Concentration for Dormancy in Starfish Blastulae

Adenosine at concentrations greater than 6 μg/ml halted embryonic development of the starfish Asterina pectinifera specifically at the 256-cell stage which corresponds to the onset of blastulation. When a fertilized egg was cultured continuously in sea water containing adenosine from fertilization, a gradual increase in intracellular concentrations of free adenosine was observed before a cessation of development took place. On the other hand, intracellular concentrations of ATP, ADP and AMP in the embryo cultured in sea water containing adenosine were nearly the same as those of an embryo cultured in sea water without adenosine. By returning the development-arrested embryo to normal sea water the embyro developed normally to the bipinnaria stage accompanied by a gradual decrease in the intracellular cencentration of adenosine.
Treatment of fertilized eggs with 9-β-d-arabinofuranosyl-9H-purine-6-amine (25 μg/ml) or 2'-deoxyadenosine (10 μg/ml) halted development specifically before the onset of blastulation in an irreversible manner. Embryos treated with 3'-deoxyadenosine (50 μg/ml) shortly after fertilization developed to healthy blastulae but hatching never occurred. These results exclude the possibilities that the action of adenosine is mediated by the inhibition of DNA synthesis or RNA polyadenylylation.     

PURIFICATION OF GONAD-STIMULATING SUBSTANCE OBTAINED FROM RADIAL NERVES OF THE STARFISH, ASTERIAS AMURENSIS

Purification of starfish gonad-stimulating substance (GSS), which induces shedding of gametes and oocyte maturation, was carried out using lyophilized radial nerves of Asterias amurensis as source material. In the first series of experiments, 1.3 mg of the purified GSS, which induced spawning at a concentration of 0.0096 μg/ml, was isolated from acetone powder of lyophilized radial nerves of 7,360 starfish through several steps of purification procedures consisting of gel-filtrations on Sephadex G–50 and G–25 columns of various sizes and ion-exchange chromatography on DEAE-Sephadex columns by gradient as well as step-wise elution. With this sample, the molecular weight and amino acid composition of GSS were estimated. Another series of experiments, conducted on a similar amount of material with purification procedures which were essentially the same as those of the first series except for employing 2 steps of partition chromatography instead of extensive gel-filtration, gave about 0.1 mg of purified sample which served as material for studies of the amino acid composition and electrophoretic properties of GSS.
The molecular weight of Asterias GSS was found to be about 2,100, as determined with the sedimentation equilibrium method. GSS seemed to consist of the following 22 amino acid residues: aspartic acid (2), threonine (1), serine (6), glutamic acid (1), proline (1), glycine (4), alanine (2), valine (1), isoleucine (1), leucine (1), histidine (1), and ornithine (1). The isoelectric point of GSS was found to be at about pH 4.5 as determined by the isoelectric focusing method.     

Regulation of Sperm Motility in Starfish. I. Initiation of Movement

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. Spermatozoa either did not move or swam with decreased beat frequency in isolated seminal plasma or in the presence of certain fractions of seminal plasma obtained by gel-filtration or by partitions. In normal sea water (containing calcium ions), a low pH of less than 6 resulted in a decrease in the number (%) of swimming spermatozoa, though the speed of propulsion and the beat frequency was not affected. On the other hand, in a calcium-free sea water, a large proportion of spermatozoa moved in both low and high pH conditions, but in low pH the speed of propulsion was reduced and many spermatozoa (31 %) swam in smaller circles.     

Dihydrofolate Reductase in Starfish Oocytes and Embryos: Developmental Consequences of Its Inhibition by Methotrexate1

Dihydrofolate reductase in immature oocytes of the starfish, Asterina pectinifera, is estimated to be 12 pg per oocyte. After completion of meiosis, the quantity of the enzyme is approximately 20 pg per egg. The content of the enzyme in the egg is kept nearly constant at this value from fertilization to the beginning of blastulation. Methotrexate, an analogue of dihydrofolate, at 20 μM did not affect meiotic maturational process and fertilization, but inhibited embryonic development at the 512-cell stage which corresponds to the beginning of blastulation. Incorporation of externally supplied deoxy[³H]uridine into DNA of the embryos cultured in the continuous presence of 20 μM of methotrexate stopped at the 256-cell stage, suggesting that the cessassion of development of the embryo at the 512-cell stage was caused by inhibition of DNA synthesis at the preceding stage. Uptake of [³H]methotrexate was low at early cleavage stages but increased just before blastulation. Externally supplied 1 mM of thymidine counteracted the inhibitory effect of methotrexate at 20 μM, suggesting that the starvation of the methotrexate-treated embryo for thymidine nucleotides halted DNA synthesis at the beginning of blastulation.     

Light-induced changes of b-type cytochromes in the electron transport chain of Euglena chloroplasts

Light-induced changes of b-type cytochromes in Euglena chloroplastswere studied spectrophotometrically.

1. In the dark and at pH 6.5, most of the cytochrome 558 in chloroplastswas in the reduced state, and most of the cytochrome 563, inthe oxidized state. Illumination of chloroplasts at pH 6.5 induceda rapid, but slight oxidation of cytochrome 552 and cytochrome558. The magnitude of photooxidation of cytochrome 558 was greatlyenhanced by the addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea(DCMU). The rate of photooxidation in the presence of DCMU wasstimulated by the addition of 0.15 µM Euglena cytochrome552, or 100 µM methyl viologen.
2. Euglena chloroplasts,incubated at 55°C for 5 min showedno significant absorbancechanges for about 10 min after theonset of illumination. However,greater photooxidation of cytochrome558 was observed afterprolonged illumination, or in the presenceof DCMU or ethylenediaminetetraaceticacid (EDTA). Similar resultswere obtained with chloroplastspre-treated at pH 9.0–10.0for 5 min.
3. At pH 9.5, andin the dark, both cytochrome 563 and cytochrome558 were inan almost reduced state. On illumination at thispH, both cytochromeswere photooxidized, with a complicatedkinetics, showing aninitial rapid and small absorbance decrease,followed by a stagnantphase of temporary retarded reaction.In the presence of DCMUor EDTA, photooxidation proceeded rapidlywithout a stagnantphase.
4. At pH 6.5 cytochrome 558, on cessation of illumination,wasquickly reduced to the initial level. At pH 9.5, there wasalsoappreciable re-reduction of cytochrome 558 and 563 whenthelight was turned off at an early stage of illumination.Theamounts of re-reduction of the cytochromes in the subsequentdark period, however, decreased as photooxidation of cytochromesproceeded. This decrease was accelerated by the presence ofDCMU.
5. At pH 9.5 ascorbate and manganese served as electrondonorsfor die DCMU-sensitive photooxidation of cytochromes558 and563.
6. Experimental results are discussed with specialreference tothe occurrence of two pools of electron carriers,one at thereducing side and the other at the oxidizing sideof photosystem2. The role of manganese in the latter pool ofelectron carriersis also discussed.

(Received March 11, 1970; )     

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.