CORTICAL AND SUBCORTICAL CHANGES PRECEDING FURROW FORMATION IN THE CLEAVAGE OF NEWT EGGS

In the first cleavage of the egg of the newt, Cynops(Triturus) pyrrhogaster, some sort of preparation for the furrow formation in the cortical and subcortical cytoplasm precedes the advancing tip of the cleavage furrow. This is shown by the following facts: (1) Incisions made close to the tip of the cleavage furrow do not stop the progress of furrowing, allowing the furrow to cross the incisions and appear on the farther sides, while incisions made far enough from the furrow tip always prevent the further travelling of the furrow, (2) Displacement of the subcortical cytoplasm ahead of the furrow by rubbing with a hair loop makes the furrow bend corresponding to the width of the rubbed area, and (3) Transplantation of the subcortical material of the furrow tip to lateral parts in the same egg causes a depression in the overlying cortex at the transplanted position. The linear extension of the prepared area for the furrow formation is the longest in the animal hemisphere and it decreases in gradient towards the vegetal pole.     

INSERTION OF STRIPS OF CELLOPHANE OR POROUS FILTERS IN THE FUTURE COURSE OF THE ADVANCING CLEAVAGE FURROW OF THE NEWT EGG *

In the area between the equatorial and the upper vegetal regions of the egg of the newt, Cynops (Triturus) phrrhogaster, strips of cellophane, ordinary filter paper or Millipore filter respectively were inserted transversely at short distances ahead of the advancing cleavage furrow. When a cellophane strip is inserted across the future path of the furrow at 0.5–0.7 mm beyond the furrow tip, the furrow stops on reaching the position of the inserted cellophane strip. If, however, a strip of the Millipore filter is inserted at the same distances ahead of the cleavage furrow, the furrow reaches the one side of the Millipore filter strip and soon appears on the opposite side of it, just as the furrow jumps across the strip, to continue the previous course. Similar tendency is observed in the experiments on insertion of strips of the ordinary filter paper. It is suggested that certain propagating factors which are necessary to induce formation of the cleavage furrow can go through the pores of the Millipore filter to precede the advancing tip of the cleavage furrow.     

微丝在卵子极性产生、分裂沟形成和极体排放中的作用

小鼠生发泡（ＧＶ）期卵母细胞在１μｇ／ｍｌ细胞松弛素Ｂ（ＣＢ）中培养,部分微丝解聚,卵母细胞不 能产生极性而在细胞中部形成分裂沟（假分裂）;极泡期印在１μｇ／ｍｌ ＣＢ中,分裂沟继续收缩,排放第 一极体。假分裂的分裂沟和极泡期的极区分裂沟形成均与分裂器中体位置相关。部分微丝解聚并不影响 假分裂和第一极体排放;全部微丝解聚（１０μｇ／ｍｌ ＣＢ）将中断假分裂和胞质分裂,分裂沟消失,卵恢 复球形。由此可见,成熟过程中卵母细胞极性的产生及分裂沟的形成都依赖于微丝的聚合。胞质分裂和 第一极体排放同样需一定量的微丝存在。     

EFFECTS OF BENZIMIDAZOLE ON CLEAVAGE OF THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins were treated with benzimidazole. They were fertilized after being kept in normal sea water for a certain period. It was found that the first cleavage occurred much earlier than in the control. The eggs had a tendency to cleave directly into 3 or 4 cells. Benzimidazole induced some visible changes in unfertilized eggs, which was considered to be the result of an insufficient activation. Benzimidazole was found to have the same effect as hypertonic solution has in Loeb's “double treatment” method for artificial parthenogenesis. When eggs activated with butyric acid were treated with benzimidazole instead of hypertonic solution, they cleaved in a high percentage.     

EFFECT OF DNP ON ACCELERATION OF THE CLEAVAGE FOLLOWING INSUFFICIENT PARTHENOGENETIC ACTIVATION IN THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, were treated with 4–5% butyric acid-sea water for 40–60 sec so that they were activated partheno-genetically without visible cortical changes. When these insufficiently activated eggs were inseminated 90–120 min after butyric acid-treatment, they divided much earlier than the control eggs in the first cleavage cycle. In the present paper, it becomes clear that if eggs are put into m /2,000-m /16,000 DNP-sea water at 60 min after insufficient activation and 30 min later, returned to normal sea water and then inseminated, they still show acceleration of the first cleavage in the same degree as the eggs which are not treated with DNP, while if eggs are exposed to DNP for 30 min prior to the insufficient activation or within 60 min after the activation, they do not show any acceleration of the cleavage. From these results, it may be concluded that some preparations for cleavage acceleration which are arrested by DNP become ready in the eggs at an early period in the first cleavage cycle and these preparations cannot be cancelled by DNP-treatment once they have been completed.     

多聚赖氨酸引起林蛙卵表面的聚集以及对分裂沟的影响

1.多聚赖氨酸能使经异硫氰基荧光素染色的卵表面均匀分布的荧光聚集成点状或块状的斑块,荧光聚集时膜下的色素颗粒随之同步聚集。早期的聚集有三种方式。2.聚集的迟早和出现斑块的大小在卵表面不同区域是不同的。腹方新月区最早;灰色新月区次之;动物性半球稍迟,荧光和色素颗粒最后都集中到动物性极形成顶帽,其余区域成为失去色素颗粒的区域;聚集引起收缩,最后植物性半球常常破裂。3.未受精卵亦出现与上述相同的反应,这提示腹方新月区可能是精子进入卵子的区域。4.细胞松弛素不能抑制多聚赖氨酸引起的聚集。5.多聚赖氨酸能抑制分裂沟的出现,能使已出现的分裂沟消失。6.失去色素颗粒区域的细胞膜在形态、功能等方面与分裂沟中的新膜相同。对新膜出现的位置和形成因素进行了讨论。     

SPERM PENETRATION AND THE FORMATION OF A FERTILIZATION CONE IN THE COMMON CARP EGG*

Sperm penetration and the formation of a fertilization cone in the micropylar canal of the egg of the common carp were examined by electron microscopy. The overwhelming majority of inseminated eggs fixed without immersion in fresh water showed that the first spermatozoon had penetrated into the ooplasm before the cortical reaction had occurred, and in many cases had formed a fertilization cone to plug the micropylar canal. At this stage the sperm head was usually located at the base of the cone, and the tail part did not participate in the formation of the cone. Inseminated eggs fixed soon after immersion in fresh water showed that the elevation of the fertilization membrane and the simultaneous recession of the fertilization cone often permitted the penetration of a few supernumerary spermatozoa into the perivitelline space near the micropylar canal, but polyspermic fertilization was never observed. The mechanism of the block to polyspermy in the egg of the common carp is discussed in connection with the fertilization cone.     

CLEAVAGE FURROW FORMATION IN A TELOLECITHAL EGG (LOLIGO PEALII) : I. Filaments in Early Furrow Formation

Formation of the first cleavage furrow in the telolecithal egg of Loligo was studied with the electron microscope. Before the actual furrow forms, a dense filamentous band develops below the plasma membrane from membrane-bounded dense bodies which appear to be Golgi-derived. The egg surface is thrown into a number of longitudinal folds which parallel the furrow and eventually become incorporated into it. These longitudinal folds contain a network of tubules and vesicles. Frequently, multivesiculate bodies are associated with the furrow and possibly give rise to the network of tubules and vesicles. Apparently part of the membrane between the two new blastomeres is derived from the surface of the longitudinal folds. The theory of furrow formation by contraction is discussed in light of the filamentous band.     

INDUCTION OF A FURROW-LIKE DENT ON THE SURFACE OF THE NEWT EGG BEFORE THE ONSET OF CLEAVAGE*

Sawai (2) found in the amphibian egg that furrow-inducing cytoplasmic component (FIC) was localized along the cleavage furrow, which could induce a furrow on the polar surface of cleaving egg under which FIC was injected. But this procedure failed on the surface of uncleaved fertilized egg. In the present experiments, an attempt was made to induce a cleavage furrow on the surface of uncleaved egg of the newt, Cynops pyrrhogaster. A piece of the cortex was cut from the uncleaved egg, which was transplanted to the egg just before or just after the onset of the cleavage, using a fine glass needle. After the transplantation FIC was injected beneath the graft with a capillary. The graft reacted to FIC and a furrow-like dent was induced at the position. Besides, stiffness of the graft increased during the cleavage of the host egg. In contrast to the cortical grafting, a large amount of the cytoplasm excluding FIC was injected under the cortex of an uncleaved egg. After several minutes FIC was deposited at the site. A furrow-like dent was formed there in many cases.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS 1. THE INHIBITION OF MICROMERE FORMATION AT THE FOURTH CLEAVAGE*

Eggs of the sea urchins, Hemicentrotus pulcherrimus, Temnopleurus toreumaticus and Pseudocentrotus depressus were used as materials. Embryos were exposed to the surfactants such as SLS, CTAB, digitonin, Tween 80, sodium deoxycholate and Lubrol P. If embryos are kept in the solutions of SLS, CTAB and digitonin, 4 vegetal cells of the 8-cell stage divide equally at the fourth cleavage and consequently 16 equal-sized blastomeres are formed at the 16-cell stage. In this case, micromere formation is inhibited by the equal cleavage. The minimum effective concentration of the surfactants for the equal cleavage gradually increases as the time performing the treatment is postponed. The continuous exposure to the surfactant is unnecessary for the inhibition of micromere formation. In the egg temporarily exposed during the earlier stage, the equal cleavage occurs at the fourth division in natural seawater. Micromere formation is strongly affected by the surfactant (SLS) at the mid 4-cell stage.     

CYTOPLASMIC DYNEIN OF THE SEA URCHIN EGG I. PARTIAL PURIFICATION AND CHARACTERIZATION*

Cytoplasmic ATPase of sea urchin eggs was partially purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel-filtration chromatography and sucrose density gradient centrifugation. The specific activity increased to 0.7 μmole/min/mg protein indicating 100 fold purification. The ATPase had a sedimentation constant of 12S and was highly specific for ATP. The enzyme fraction contained neither (Na, K)-ATPase, Ca-ATPase, oligomycin-sensitive ATPase, phosphatases, nor myosin. This cytoplasmic ATPase was inhibited by a low concentration of vanadate (V). Half-maximal inhibition was observed at a vanadate concentration of 1 μM at low ionic strength. The inhibition was almost totally reversed by addition of norepinephrine. The vanadate-sensitivity of cytoplasmic ATPase decreased with increasing KCl concentration. The activation by Mg²⁺ or Ca²⁺, and dependence of the activity on KCl concentration characteristic of dyneins from sea urchin sperm flagella and the embryonic cilia were observed with cytoplasmic ATPase. These results allowed the cytoplasmic ATPase to be classified as a dynein. In addition, this designation was reinforced by the fact that an oligomeric 23S form of cytoplasmic dynein was identified in the cytoplasm as well as in the isolated mitotic apparatus.     

AN ELECTRON MICROSCOPIC STUDY OF THE DEVELOPMENT OF THE CLEAVAGE FURROW IN MAMMALIAN CELLS

The process of cytoplasmic cleavage has been studied in thin sections of rat erythroblasts and the cells of mouse leukemia and Walker 256 carcinoma of the rat. The development of the cleavage furrow begins in relation to the mid-body, which, earlier, appears on the equatorial plane in association with the continuous fibers of the spindle. The earliest evidence of a cleavage furrow is the presence of a vesicle or vesicles close to the mid-body. Subsequently, many smaller vesicles are seen in the equatorial plane. The cleavage furrow probably develops by the fusion of these vesicles so that a new plasma membrane is formed between the daughter cells, and extends from the telophase intercellular bridge to the cell margin. During the stage of formation of the vesicles, cisternae, believed to be part of the endoplasmic reticulum, assume an intimate relationship with the cleavage plane, and they may perhaps be involved in the formation of the vesicles.     

AN EXPERIMENTAL ANALYSIS OF THE ROLE OF CYTOPLASMIC FOUNTAIN STREAMING IN FURROW ESTABLISHMENT

During furrow establishment the equatorial cell surface is altered so that it becomes capable of exerting the physical force required for cytokinesis. It has been suggested that the alteration results from deposition of a furrow-organizing substance at the equator by convergence of “fountain streaming” patterns of cytoplasmic flow. The purpose of this investigation was to determine whether a fountain streaming pattern is essential for furrow establishment. The shape of sand dollar eggs was altered before the time of furrow establishment in ways that would greatly disrupt fountain streaming. Shapes that should have abolished regions of convergence and shapes that should have created several such regions were created by notching and perforating cells. Furrow establishment took place under circumstances that would be highly unlikely if a fountain streaming pattern of cytoplasmic currents were necessary.     

PARTICIPATION OF THE CYTOPLASMIC MEMBRANE IN THE GROWTH AND SPORE FORMATION OF BACILLI

A polyester embedding technique was used to study the early stages of spore formation in members of the genus Bacillus in order to investigate further the origin and nature of the initial spore septum and the resulting forespore envelope. Whereas previously, with a methacrylate procedure, this layer had appeared to be continuous with the cell wall, this study reveals it as a double layer of cytoplasmic membrane. Perisporal, membranous organelles connected both to the developing forspore envelope and to the cytoplasmic membrane were encountered in the four species studied. Similar organelles were prominent during growth at the sites of transverse septa formation. These were connected to, or continuous with, the cytoplasmic membrane and often adherent to the chromatin bodies of the dividing bacilli.     

EXPERIMENTAL STUDIES ON THE SITE OF PROPAGATION OF THE FERTILIZATION-WAVE IN THE SEA URCHIN EGG

Although the fertilization-wave in the sea urchin egg is generally considered to propagate over the egg surface, there has been no definite evidence to show the site of propagation. The possibility that the wave passes through the endoplasm, not over the egg surface, has not been denied.
A drop of paraffin was injected into an egg, so that the endoplasm was divided into 2 parts by the paraffin drop, the 2 parts being connected only by the egg cortex. When spermatozoa were added to one side of the egg, the fertilization membrane was formed first on this part of the egg and then on the opposite part. This indicates that the egg surface or the egg cortex is the site of propagation of the fertilization-wave and the endoplasm has no direct influence on the propagation.     

INDUCTION OF ASTER FORMATION AND CLEAVAGE IN EGGS OF THE SEA URCHIN HEMICENTROTUS PULCHERRIMUS BY INJECTION OF SPERM COMPONENTS*

Studies were made on which components of sperm were able to induce aster formation and cleavage of eggs of the sea urchin Hemicentrotus pulcherrimus. The sperm components were separated by homogenization and centrifugation into the following 3 fractions: the head-midpiece, midpiece and tail. The head-midpiece fraction was then divided into 2 sub-fractions, the centriole sub-fraction and the centriole-free sub-fractions. Each fraction was injected into unfertilized eggs and after 15–30 min the eggs were inseminated. The ability of a fraction or a sub-fraction to induce aster formation and cleavage was deduced from the frequency of multipolar cleavage. The head-midpiece fraction and the centriole sub-fraction were effective in inducing aster formation and cleavage, but the other fractions were not. It was concluded that isolated centrioles from sea urchin sperm act as division centers in the egg.     

CHANGES IN THE APPARENT CYTOPLASMIC HYDROGEN ION CONCENTRATION OF AMEBA DUBIA ON INJECTION OF EGG ALBUMIN

1. Egg albumin when injected into an ameba or discharged into the solution about it raises the apparent pH of the cytoplasm of the ameba. 2. With time the cytoplasm returns to the original pH 6.9 if the nucleus is present. Amebae that have received repeated injections of albumin in some cases extrude their nuclei. In these cells the cytoplasm remains at the more alkaline pH induced by the albumin for at least 12 hours. 3. When a 2 per cent solution of albumin is introduced into a suspension of amebae there is a temporary marked rise in the rate at which CO₂ is given off with no corresponding rise in O₂ uptake. 4. The results observed can be explained if the albumin discharged onto the surface of the ameba rapidly enters the cell and there becomes distributed in a phase of the cytoplasm other than the one which contains the phenol red.     

ON THE DE NOVO FORMATION OF THE CENTRIOLE IN THE ACTIVATED SEA URCHIN EGG

Eggs of Pseudocentrotus depressus were activated artificially by Loeb's "double treatment method". 50 min after activation, a number of asters were produced in the eggs. It was confirmed by electron microscopy that centrioles with a typical fine structure were present in artificially induced asters.
An unfertilized egg of Hemicentrotus pulcherrimus was divided into 2 halves, nucleated and non-nucleated, by centrifugation on a sucrose bed. Each half was activated by the same method as mentioned above. Several asters were produced in both halves after a certain period of incubation. The presence of bodies considered to be centrioles were demonstrated in the asters in both nucleated and non-nucleated halves.
The results add probability to the view that the centrioles are produced de novo in artificially activated eggs and fragments.     

THE GOLGI ZONE OF THE RAT SPERMATID AND ITS ROLE IN THE FORMATION OF CYTOPLASMIC VESICLES

Electron microscopic study of the Golgi zone of the rat spermatids revealed two main types of vesicular structures: flat vesicles generally accumulated in stacks and spheroidal vesicles of various sizes. The Golgi zone of young spermatids showed a predominance of flat vesicles and small spherical vesicles, while the "residual" Golgi zone of the maturing spermatids displayed an increased number of large spherical vesicles and thus became increasingly vacuolated in appearance. The images presented were suggestive of a transformation of the flat vesicles into spherical vesicles by fragmentation and dilatation and also of a passage of the spherical vesicles from the Golgi zone to the surrounding cytoplasm.