Hiroshi Imoh

Accumulation of Annulate Lamellae in the Subcortical Layer during Progesterone-induced Oocyte Maturation in Xenopus Laevis

Changes in the fine structure, the location and the number of stacks of annulate lamellae during progesterone‐induced maturation of oocytes of Xenopus were determined by electron microscopy. longitudinal sections of full‐grown oocytes, about 260 stacks of annulate lamellae were observed with marked concentration in the subcortical layer, particularly in the vegetal hemisphere. After exposure to. progesterone, annulate lamellae increased and accumulated further in the subcortical layer. A significant increase of annulate lamellae around the vegetal side of the germinal vesicle seen 3 h after progesterone application. In oocytes 6 h after progesterone application, an average of 320 stacks of annulate lamellae were seen in longitudinal sections and more than two‐thirds of the pore complexes of annulate lamellae were localized in the subcortical layer less than 50 from the oocyte surface, the rest being distributed in the deeper ooplasm. At the time of ger‐ minal vesicle breakdown, all the annulate lamellae underwent complete decomposition. The results were discussed from the view point of comparative developmental biology.

Distribution of the Germinal Vesicle Material during Progesterone‐Induced Oocyte Maturation in Xenopus and in Cynops

The distribution of the germinal vesicle material in the oocyte during progesterone‐induced maturation was studied in Xenopus and in Cynops. In both species, two distinctive masses of yolkfree cytoplasm appear in specific areas of the oocyte and at definite stages of maturation. One, the primary cytoplasmic mass, is formed at the basal side of the germinal vesicle during early maturation and is very RNA‐rich. In Xenopus, a large part of the primary cytoplasmic mass persists as a mass during maturation and ends up as a thin disk at the boundary between the animal and the vegetal hemisphere in the mature oocyte. In Cynops, a rod‐like primary cytoplasmic mass extends near to the equatorial zone and becomes indistinct in the mature oocyte. The other, the secondary cytoplasmic mass, is formed at or prior to germinal vesicle breakdown in areas around the germinal vesicle and is also RNA‐rich. The secondary cytoplasmic mass is dispersed and constitutes the RNA‐rich animal hemisphere cytoplasm in the mature oocyte. Observed results suggest that the primary and the secondary cytoplasmic mass contain different germinal vesicle materials.

Appearance and Distribution of RNA‐Rich Cytoplasms in the Embryo of Xenopus laevis during Early Development

The occurrence of the dorsal yolk‐free cytoplasm in the fertilized egg of Xenopus was re‐examined, and the appearance and the distribution of RNA‐rich cytoplasms in Xenopus embryos during early development were examined with their paraffin sections. The results show that the dorsal yolk‐free cytoplasm does not occur solely in the dorsal part of the embryo but is continuous to similar cytoplasmic mass in the central and the ventral part. The whole mass of this continuous cytoplasm is denoted here as the mesoplasm. The locations of the mesoplasm in the embryo can be traced by its high RNA content during cleavage and blastulation. The cells endowed with the mesoplasm constitute a broad band about the equator of the blastula. At the lower edge of this band, the blastopore lip is formed during gastrulation. Another mass of yolk‐poor and RNA‐rich cytoplasm becomes distinct around every nucleus in the stage 4 embryo and is denoted here as the nucleophilic plasm. This plasm is diminished at every nuclear division and disappears in the stage 10 embryo. Origins and roles of the mesoplasm and the nucleophilic plasm were discussed and a mechanism of blastulation was suggested.

Formation of the Neural Plate and the Mesoderm in Normally Developing Embryos of Xenopus laevis

Normally developing embryos of Xenopus were fixed at various stages between the blastula and early tail bud stage, and their serial sections were examined. The marginal belt of the blastula was characterized by abundance of cells with RNA‐rich peripheral cytoplasm called mesoplasm. At the early gastrula stage, the marginal belt was folded into two layers giving rise to mesodermal material and marginal ectoderm. During gastrulation, the mesodermal material, which consisted of RNA‐rich cells, spread to enclose the blastocoel and the endoderm, and a large part of it was shifted to the dorsal side of the embryo. It gradually established the mesodermal layer. The notochord was formed on the dorsal lip of the blastopore by involution, separately from preformed mesodermal material. The RNA‐rich cells in the marginal ectoderm became columnar, forming a broad belt in the marginal zone. This belt was deformed and shifted to the dorsal side during gastrulation, eventually establishing the neural plate showing quantitative differentiation along the head‐tail axis. Possible mechanisms involved in the formation of the neural plate and mesoderm were discussed with reference to the organizer and the mesoplasm.

Direct evidence of an essential role for extended involution in the specification of a dorsal marginal mesoderm during Cynops gastrulation

It has been indicated that specification of the dorsal marginal mesoderm of the Cynops gastrula is established by vertical interactions with other layers, which occur during its extended involution. In the present study, when the prospective notochordal area of the early gastrula was almost completely removed together with the dorsal mesoderm‐inducing endoderm and most of the bottle cells, the D‐less gastrulas still formed the dorsal axis with a well‐differentiated notochord; in half of them, where the involution occurred bi‐laterally, twin axes were observed. On the other hand, when the wound of a D‐less gastrula was repaired by transplanting the ventral marginal zone and ectoderm, the formation of the dorsal axis was inhibited if the involution of the lateral marginal zone was prevented by the transplanted piece. The present study suggests that: (i) cells having dorsal mesoderm‐forming potency distribute farther laterally than the fate map; and (ii) the extended involution plays an essential role in the specification of the dorsal marginal mesoderm, especially in notochordal differentiation in normal Cynops embryogenesis.

POSSIBLE SIGNIFICANCE OF NUCLEAR VOLUME CHANGE DURING EARLY DEVELOPMENT OF NEWT EMBRYOS

The nuclear volumes at several stages of development were measured on Triturus pyrrhogaster embryos and changes in the fine structure and reactivity towards alkaline fast green of the nuclei were also examined. It was shown that the blastula nuclei were reduced about 80% to reach a constant volume of about 1,400 μm3 by the tail bud stage in ectomesodermal parts of the embryos. In the endoderm, the decrease in the nuclear volume was slightly delayed.

CHANGES IN NUCLEOLI AT MATURATION OF NEWT OOCYTE

Changes in the nucleoli of maturing oocytes and the eggs of Cynops pyrrhogaster were studied with light and electron microscopy. Extrachromosomal nucleoli moved toward the center of the germinal vesicle in response to the maturation stimulus, released presumed ribosomal ribonucleoprotein, and further moved toward the center of the nucleus to form an aggregate with chromosomes which behaved in a similar manner. A few. ball‐shaped nucleolar masses were formed from this aggregate, leaving the chromosomes and probably the extrachromosomal nucleolar organizer. The chromosomes then proceeded to the first meiotic metaphase. The nucleolar masses were surrounded by a layer of mitochondria and became smaller with formation of pinched‐off fragments, which were also surrounded by mitochondria, during the time the egg was moving down the oviduct. Only fragments were observed in the subcortical area of the animal hemisphere of the egg after reaching the lowest part of the oviduct.

Study of Cynops pyrrhogaster notochord differentiation using a novel monoclonal antibody

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole‐mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior–posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm‐inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.

OCCURRENCE AND FINE STRUCTURE OF SUBCORTICAL CYTOPLASMIC ISLETS IN FERTILIZED EGGS OF CYNOPS PYRRHOGASTER

Fertilized and unfertilized eggs of Cynops pyrrhogaster were examined by light and electron microscopy. In fertilized eggs that have just been laid, there are numerous small cytoplasmic patches free of granules in the pigmented layer of the animal hemisphere. Many of these granule‐free cytoplasmic islets gradually grow out subcortically from the pigmented layer and fuse to form a subcortical layer of yolk‐free cytoplasm of varying thickness by the time of the first cleavage division. The cytoplasmic islets are present in 100% of the fertilized eggs, but not in unfertilized eggs. Electron microscopic observations showed that the cytoplasmic islets contain tubules and that development of a complex system of cortical tubules constitutes the basis of the early growth of the cytoplasmic islets. The cortical tubules are transient structures and are no longer observable a few hours after the eggs are laid. These phenomena are considered to be a response of the egg to the fertilization stimulus.