Shin-ichi Nemoto

MARKED DECREASE IN THE RIGIDITY OF STARFISH OOCYTES INDUCED BY 1‐METHYLADENINE1

Extreme rigidity of immature starfish oocytes as measured by compression method was found to decline during the early phase of their maturation when induced by 1‐methyladenine (1‐MeAde). The onset of this decrease in stiffness occurred within 5 to 9 min of 1‐MeAde treatment, well before the breakdown of the germinal vesicle, progressively declining to reach a minimum stiffness after 20 min. Dithiothreitol, known as an artificial maturation‐inducing agent, caused a similar change. The stiffness is thus expected to serve as a quantitative indicator of the early process of cytoplasmic events, which would induce the breakdown of the germinal vesicle. Cytochalasin B (3 μg/ml) also reduced the stiffness, but unlike the former two agents, the effect was reversible, and did not interfere with the process of maturation. Due to the effect of cytochalasin B, it became possible to enucleate immature oocytes by centrifugal force. Non‐nucleate fragments thus obtained still maintained their marked stiffness, which was decreased by the action of 1‐MeAde, with a time‐course similar to that of intact oocytes.

Ultrastructural Studies on the Behavior of Centrioles during Meiosis of Starfish Oocytes

The behavior of centrioles and ultrastructural changes of the nucleus were observed in maturing oocytes of the starfishes, Asterina pectinifera and Asterias amurensis. Observations were focused on the number and behavior of centrioles during two successive meiotic divisions. Examination of serial sections revealed that in meiosis I each division pole has a pair of centrioles, whereas in meiosis II each has only one centriole, confirming the observations by Sluder et al. (1989) on oocytes of Pisaster ocraceus and Asterias forbesi. The first polar body had two centrioles and the second polar body had only one. These results indicate that no duplication of centrioles occurs during the two successive meiotic divisions, and that the egg inherits one centriole from a primary oocyte.

PERIODIC CHANGES IN THE CONTENT OF PROTEIN‐BOUND SULFHYDRYL GROUPS AND TENSION AT THE SURFACE OF STARFISH OOCYTES IN CORRELATION WITH THE MEIOTIC DIVISION CYCLE*

The sulfhydryl content of protein and the tension at the surface were measured for starfish oocytes from the first meiotic division to the cleavage stage.

Nature of the 1‐Methyladenine‐Requiring Phase in Maturation of Starfish Oocytes

Reinitiation of meiosis in starfish oocytes requires the continuous presence of 1‐methyladenine (1‐MeAde) in the surrounding medium for a definite period. The length of the ‘hormone‐dependent phase’ (HDP) in Asterina pectinifera, which was defined as the time necessary for induction of 50% germinal vesicle breakdown (GVBD), was found to be about 11 min at 17°C, and 8 min at 20°C. Repeated treatments for shorter periods with 1‐MeAde revealed that the action of this agent was cumulative, and that stable intermediate states between the unstimulated and fully stimulated levels existed during the HDP. Measurement of the stiffness of oocytes also demonstrated this stable intermediate state. Thus, there may be a factor(s) in the cytoplasm that accumulates continuously during the HDP and triggers GVBD when it reaches a critical level(s). When dithiothreitol (DTT) was used as an artificial maturation‐inducing agent, the intermediate state was far less stable, suggesting a difference in the modes of action of 1‐MeAde and DTT. Isotonic CaCl2, the Ca2+ ionophore (A 23187) and methylxanthines, which are known to cause increase in intracellular Ca2+, had additive effects with 1‐MeAde. These results suggest that part of the action of 1‐MeAde is to release Ca2+ in the oocyte cytoplasm.

Centrosome destined to decay in starfish oocytes

In contrast to the somatic cell cycle, duplication of the centrioles does not occur in the second meiotic cycle. Previous studies have revealed that in starfish each of the two centrosomes in fully-grown immature oocytes consists of two centrioles with different destinies: one survives and retains its reproductive capacity, and the other is lost after completion of meiosis. In this study, we investigated whether this heterogeneity of the meiotic centrioles is already determined before the re-initiation of meiosis. We prepared a small fragment of immature oocyte containing the four centrioles and fused it electrically with a mature egg in order to transfer two sets of the premeiotic centrioles into the mature cytoplasm. Two asters were present in this conjugate, and in each of them only a single centriole was detected by electron microscopy. In the first mitosis of the conjugate artificially activated without sperm, two division poles formed, each of which doubled in each subsequent round of mitosis. These results indicate that only two of the four premeiotic centrioles survived in the mature cytoplasm and that they retained their reproductive capacity, which suggests that the heterogeneity of the maternal centrioles is determined well before re-initiation of meiosis, and that some factor in the mature cytoplasm is responsible for suppressing the reproductive capacity of the centrioles destined to decay.

Manipulative methods for analyzing embryogenesis

Publisher Summary This chapter describes a number of manipulative methods adopted to analyze the egg cells and embryos of the sea urchin Hemicentrotus pulcherrimus and the starfish Asterina pectinifera . With minor modifications, they will be applicable to other species. By using egg fragments, the possible roles for the nucleus in cytoplasmic activities or, in turn, possible roles for cytoplasmic materials in embryogenesis are examined. There are two ways to obtain egg fragments: manual bisection and centrifugal separation. Manual bisection enables to divide eggs in any desired direction so that single egg fragment can be compared with its complementary partner. On the other hand, centrifugal separation makes large enough numbers of nonnucleate and nucleate egg fragments for biochemical analyses. The chapter describes how horseradish peroxidase (HRP) is injected into blastomeres of starfish embryos through the intact fertilization membrane. Chromosome preparations of starfish embryos are explained.

Changes in cGMP levels on meiosis reinitiation of starfish oocytes

An intracellular level of cGMP (but not cAMP) transiently decreased the reinitiation of oocyte maturation in the starfish, Asterias amurensis. Exogenously applied cGMP inhibited hormone-induced maturation. Methylxanthines inhibited oocyte maturation by suppressing the decrease in cGMP levels. These results suggest that a decrease in cGMP levels is a prerequisite for meiosis reinitiation of starfish oocytes.

Role of Specialized Microvilli and the Fertilization Envelope in the Spatial Positioning of Blastomeres in Early Development of Embryos of the Starfish Astropecten scoparius

In the eggs of a wide range of animal species, various factors that determine the blastomeres’ presumptive fate are known to locate unevenly within the egg. In the embryos of these animals, cleavage occurs not just to increase cell numbers, but also to distribute the factors to the respective blastomeres, resulting in cell specialization at the later stages. In the early cleavage stages, before the establishment of a device such as desmosomes to directly join the blastomeres, some other means is needed to keep the blastomeres together and maintain the relative positions among them. In this study, we found that the embryos of the starfish Astropecten scoparius lack the hyaline layer seen in sea urchin embryos and that blastomeres adhere to the fertilization envelope (FE) via filamentous cellular projections (fixing processes). Electron microscopy revealed the fixing processes to be specialized microvilli formed, after the elevation of the FE, by the elongation of short microvilli that pre-exist in unfertilized eggs. After the first cleavage, the two blastomeres separate from each other and finally attach to the FE. In the subsequent cleavages, the blastomeres undergo repeated cell division without separating from the FE. Between the blastomeres and the FE, only shortened fixing processes were observed. Destruction of the fixing processes caused release of the blastomeres from the FE and disturbance of the relative positions of the blastomeres, resulting in abnormal development of the embryos. These observations suggest that the fixing process is a device to keep the egg placed centrally in the FE up to the first cleavage, and after the first cleavage and beyond to anchor the blastomeres to the FE so that the FE can be used as a scaffold for morphogenesis. Electron microscopy also suggests that the inner layer of the FE, which is derived from the contents of cortical granules, reinforces the adhesion of the fixing processes to the FE. Immuno-electron microscopy, using an antibody against sea urchin hyaline layer, showed that the inner layer of the FE of starfish eggs and the hyaline layer of sea urchin eggs, which are both derived from cortical granules, contain some common elements.

A nuclear extract, prepared from mass-isolated germinal vesicles, retains a factor able to sustain a cytoplasmic cycle of starfish oocytes

The germinal vesicle (GV) of starfish oocytes contains a factor which is required to drive the cytoplasmic cycle of the meiotic division. Biochemical investigation of this factor has been difficult due to the small quantities of obtainable GV materials. To overcome this, we have developed a mass-isolation procedure for the GVs of starfish oocytes, which depends on the softening of the cortex of the oocytes by cytochalasins to enable the GVs to pass through the cortex by centrifugation. From the isolated GVs, we have prepared a soluble fraction which retains the activity to induce the cytoplasmic cycle in the meiotic division of oocytes. The factor was sensitive to both heat and papain, suggesting that it is a protein.

Fertilization-induced change in the respiratory rate in eggs of several marine invertebrates

Abstract 1. 1. Fertilization enhances the respiratory rate in eggs of sea urchin and tunicate but hardly increases it in oyster, mussel, echiuroid, polychaete and starfish. In all examined species, fertilization increased the rate under infinite stimulation by 2,4-dinitrophenol. 2. 2. In these eggs, the responses of respiration to phenazine methosulfate and 2,4-dinitrophenol suggest that fertilization releases blockages of mitochondrial respiration and its coupling to oxidative phosphorylation. 3. 3. In the former two species, the blockage of respiration is quite strong in unfertilized eggs and hence, its release by fertilization probably becomes apparent in spite of intensified ADP control by releasing the blockage in coupling to phosphorylation.