Hiroko Shirai

MECHANISM OF STARFISH SPAWNING. II. SOME ASPECTS OF ACTION OF A NEURAL SUBSTANCE OBTAINED FROM RADIAL NERVE

1. When a water extract of radial nerves of the starfish, Asterias amurensis, was locally applied to one part of an isolated whole ovary for an appropriate period and then small slits were made on the ovarian wall, intense discharge of eggs occurred only in the treated portion.2. Eggs within the nontreated part of the ovary were observed to adhere to each other and to the gonadal wall by means of the follicle layer surrounding them.3. Eggs within ligated ovarian fragments which had been treated with nerve extract lost their follicles and underwent maturation. These eggs were found to be freely movable.4. Artificial spawning could be induced without using nerve extract; ovarian fragments immersed in Mg-free sea water released their eggs after a certain interval, while those treated with Ca-free sea water for an appropriate period spawn after subsequent addition of calcium.5. Breakdown of follicles occurred within an ovary treated with calcium-free sea water.6. Treatment with contraction-inducing agents suc...

Gonad-stimulating and maturation-inducing substance

Publisher Summary This chapter describes methods for preparing and assaying gonad-stimulating substance (GSS) and maturation-inducing substance (MIS). These methods are derived from experience with selected starfish. In principle, if not in specifics, they may be useful for additional species of starfish and for studying GSS and MIS in other classes of echinoderms. Although all nervous tissues in starfish probably contain GSS, only the radial nerves are easy to dissect and handle for extracting GSS. Radial nerves are located along the ambulacral groove on the oral surface of each arm. To separate radial nerves from a soft-bodied starfish such as Asrerias arnurensis, the oral surface of the arms is dissected by cutting along the rows of tube feet with scissor. To obtain large amounts of MIS, fresh ovaries (200 mg/ml) are incubated in seawater containing GSS (500 μg dry nerve Eq/ml) for 6 hours (20°C). The medium is filtered through a mesh to remove ovarian walls and then centrifuged to remove eggs (3000 rpm for 10 minutes). Identification and use of GSS and MIS in many other animals will enable more suitable materials to be used leading to a deeper understanding of spawning and oocyte maturation.

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. Oocytes whose germinal vesicles had been shifted by centrifugation from PSPBF to the opposite surface before the commencement of germinal vesicle breakdown (GVBD) (less than 15 min after 1‐MeAde treatment), failed to form polar bodies, whereas oocytes centrifuged after commencement of GVBD (20 min after 1‐MeAde treatment) did form polar bodies where their fading germinal vesicles had reached by centrifugation. In the oocytes which failed to form polar bodies by centrifugation, an aster was observed near PSPBF of each oocyte. When inseminated, every oocyte treated with 1‐MeAde developed normally irrespectively of the mode of polar body formation including the site and the occurrence, and the animal pole of every larva was derived from PSPBF.

MECHANISM OF STARFISH SPAWNING. IV. TENSION GENERATION IN THE OVARIAN WALL BY 1-METHYLADENINE AT THE TIME OF SPAWNING

Tension generation in the starfish ovaries at the time of spawning was investigated using an isometric tensiometer. Ovarian fragments containing oocytes gradually generated tension after 1-methyladenine application; before 1-methyladenine application the ovarian walls were not under strong tension. Isolated ovarian walls did not generate tension after l-methyladenine treatment, but did after the application of jelly substance. As the hormone 1-methyladenine is known to act on oocytes surrounded by follicle envelopes to induce germinal vesicle breakdown, which inevitably results in the breakdown of follicle envelopes, it is concluded that (1) jelly substance acts directly to induce contraction of ovarian walls, (2) 1-methyladenine acts indirectly, contraction being caused as a result of the hormones action on breakdown of germinal vesicles and follicle envelopes, after which jelly substance contacts the ovarian wall.

Oocyte Competence to Maturation-inducing Hormone. I. Breakdown of Germinal Vesicles of Small Oocytes in Starfish, Asterina pectinifera*

1‐Methyladenine (1‐MeAde) is the endogenous maturation‐inducing substance (MIS) in starfish. However, small oocytes have no competence to 1‐MeAde even at the concentration of 10−5M. Furthermore, when they were injected with cytoplasm of fully‐grown (large) and maturing (1‐MeAde‐treated) oocytes, known to contain maturation‐promoting factor (MPF), they did not undergo germinal vesicle breakdown (GVBD). On the other hand, germinal vesicles (GV) of the small oocytes underwent nuclear breakdown when the small oocytes were fused with the large maturing oocytes. Therefore it is concluded that the GV of the small oocytes are capable of undergoing nuclear breakdown in the presence of the sufficient MPF, but that the small oocytes can not amplify the injected MPF. Fused cells displayed particular shape changes during the course of nuclear breakdown of both the large and the small oocytes.

Oocyte Maturation in Starfish: Sequential Changes of Oocyte Shape Induced by 1‐Methyladenine Associated with Germinal Vesicle Breakdown

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). Germinal vesicle breakdown (GVBD) occurred when the a–v diameter suddenly increased. The change of the oocyte shape occurred differentially in the animal and vegital halves i.e. when the egg‐axis diameter shortened the surface of the vegital pole side shrank and the surface of the animal pole side expanded. GVBD was suppressed under hypertonic conditions and facilitated under hypotonic conditions. Cytological examination of the process of GVBD revealed (1) separation of the nuclear membrane from the granular cytoplasmic mass, (2) depression of several parts of the nuclear membrane and the increase of amorphous matrix on the outside of the nuclear membrane, and (3) disappearance (fragmentation) of nuclear membrane. The morphological changes were not the same along the egg‐axis temporally and spacially. These observations suggest firstly that the cytoplasm, which has been in a certain quiescent state with a certain rigidity before 1–MeAde administration, not only undergoes a decrease in stiffness but also acquires a contractile property after 1–MeAde administration, and secondly that contractile or cytoskeletal components do not evenly function or are not evenly arranged in the animal and vegital halves.

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.

Assembly of a protein sharing epitopes with sperm dynein heavy chains into meiotic spindle in the prometaphase starfish oocyte

Starfish oocyte meiosis provides a good system for studying the mechanism for prometaphase chromosome movement. Since a protein sharing epitopes with sperm dynein might be a force generator for mitosis, the contribution of such a protein was assessed in this movement. Specific antibodies to heavy chains (HCs) and intermediate chains (ICs) of dynein subunits were affinity‐purified from whole antidynein serum. We confirmed that the oocytes contain several polypeptides identical to sperm dynein subunits. The anti‐HCs binding to in situ antigen was examined in the oocytes permeabilized with detergent at appropriate stages of maturation with special reference to tubulin and chromosomes, and the meiotic apparatus‐establishing process was described in terms of a force generator (oocyte dynein). Before resumption of maturation, dynein HCs were particularly associated with prophase chromosomes within the germinal vesicle (GV). After GV breakdown, there was a striking local accumulation of dynein HCs in the “fading GV” (nuclear matrix). When chromosomes were pulled toward the central area between 2 asters, dynein was accumulated at first at the presumptive equator and then moved to the poles, showing uneven localization on the meuotic spindle.