CHANGES IN ACTIVITIES OF THYMIDYLATE SYNTHASE AND DIHYDROFOLATE REDUCTASE IN SEA URCHIN EGGS AFTER FERTILIZATION

Thymidylate synthase activity in sea urchin eggs increases just after fertilization and decreases 30 min later. Then, cyclic variation in the activity occurs in association with the cleavage cycle. Dihydrofolate reductase activity in fertilized eggs is almost the same as in unfertilized eggs and shows no marked change within 3 hr after fertilization. Aminopterin, an analogue of dihydrofolate, inhibits dihydrofolate reductase, and arrests cleavage. On incubation in sea water containing aminopterin (20-100μM) from the time of fertilization, the development of Clypeaster and Pseudocentrotus eggs was arrested at the 32–64 cell stage, and that of Anthocidaris eggs was arrested at the morula stage. Dihydrofolate (100μM) counteracts the inhibitory effect of aminopterin on egg cleavage. Thymidine at concentrations above 10μM also prevents inhibition by aminopterin. Other deoxyribonucleosides at concentrations of 10μM to 100μM do not affect inhibition of egg cleavage by aminopterin. Deoxyadenosine at concentrations above 5 mM inhibits egg cleavage, but other deoxyribonucleosides have no effect.     

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.     

EFFECTS OF CHEMICAL REAGENTS ON THE CYCLIC CHANGES OF CORTEX AND CYTOPLASM IN THE ACTIVATED ENUCLEATED EGG-FRAGMENTS OF THE SEA URCHIN*

Unfertilized eggs of sea urchins. Anthocidaris crassispina and Hemicentrotus pulcherrimus, were separated by centrifugation into two fractions (nucleated light and enucleated heavy fragments). The enucleated egg-fragments were activated by treatment with 1 M urea and then put into sea water solutions of the following three reagents; colcemid, cytochalasin B and Monogen at a concentration by which cleavage was suppressed. It was then examined whether the egg-fragments can exhibit cyclic changes of cytoplasm and cortex in correlation with the cleavage cycle in normally fertilized eggs without any influence of nuclear activity. The results obtained clearly showed that colcemid can suppress the cyclic appearance of cytoplasmic changes, but not that of cortical changes; on the contrary, in cytochalasin B- and Monogen-treated fragments, the periodicity in cortical activities is suppressed, while the periodic changes in the cytoplasm appear according to a timeschedule of the cleavage cycle. Therefore, it may be said that: 1) cyclic changes can occur in both the cytoplasm and the cortex independently, without the direct influence of nuclear activity; 2) if either of them is arrested, the cleavage does not take place; 3) the normal cleavage requires the simultaneous occurrence of periodic activities both in the cortex and in the cytoplasm after fertilization.     

THE RELATION BETWEEN THE ELECTRICAL CONDUCTIVITY OF THE EXTERNAL MEDIUM AND THE RATE OF CELL DIVISION IN SEA URCHIN EGGS

Dilution of sea water with isotonic sugar solution leaves the rate of cleavage of Arbacia eggs almost unchanged until the proportion of sea water is decreased to 20 or 25 volumes per cent. From this point cleavage becomes progressively slower with further dilution. Many eggs fail to cleave at dilutions of 5 to 6 volumes per cent. No cleavage occurs in 2 volumes per cent sea water or in pure sugar solution. Eggs returned from these media to sea water resume cleavage and development. There is thus no relation between the rate of cleavage and the electrical conductivity of the medium, except possibly within the range of dilutions from 20 to 5 volumes per cent sea water. In this range cleavage rate decreases as conductivity decreases, but the relation is not a linear one.     

银鲫受精卵中卵黄粒和线粒体的超微结构变化

电镜观察到雌性银鲫肝脏中正在形成的卵黄物质具有晶形主体结构,银鲫卵的卵黄粒内无晶形主体,第一次卵裂前的受精卵卵黄粒内有两种形式的空泡。一种是一些中空的小空泡;另一种是一个大的空泡,泡内含有线粒体和颗粒状的核糖体,泡的边缘有片层状的类脂物。受精卵中的线粒体内部结构多样,有些含有颗粒状物,有些含有片层状的类脂物。       

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.     

ELECTROPHORETIC MOBILITY OF SEA-URCHIN EGGS DURING THE DIVISION CYCLE

The electrokinetic potential of fertilized sea-urchin eggs, without the fertilization membrane and hyaline layer, was investigated by measuring the electrophoretic mobility of the eggs from fertilization to the second cleavage. A cyclic change in mobility was found to accompany the division cycle: the peak of the change was observed about 15 min before the appearance of both the first and second cleavage furrows.
A smaller peak was observed at 20–30 min after fertilization, but such a peak was not repeated between the first and the second cleavage.
Fertilized eggs with the fertilization membrane intact did not show a significant change in electrophoretic mobility throughout the division cycle.     

A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA

A fine structural study has been carried out on the various formed elements present before, during, and after the first cleavage division, not only in normally developing Arbacia eggs, but also in eggs which have been induced to cleave prematurely by high-pressure centrifugation. The aim has been to ascertain whether or not any of the morphologically identifiable components may be involved in initiating the furrowing process. Also, attention has been given to the fine structure of the cytoplasmic cortex, particulary in the walls of the furrow, in the hope of reaching a better understanding of the mechanics of cleavage. The annulate lamellae and the membranous envelope of the nucleus are the only formed elements which disappear shortly before cleavage, not only in eggs undergoing normal division, but also in eggs which have been induced to cleave ahead of schedule by high-pressure, high-force centrifugation. Therefore, it is suggested as a tentative hypothesis that materials liberated upon disintegration of the nuclear membrane and the annulate lamellae play an essential role in initiating and effecting the furrowing reaction, especially since the stratification of these elements in experimentally induced eggs corresponds to the position of the developing furrow. Another of the membranous elements in the egg, the Golgi complex, shows considerable modification as a result of high-pressure centrifugation, but these structures do not undergo disintegration. Rather, they become curled into rounded bodies. The vacuole population is not greatly affected by inducing treatments. During cleavage, both naturally occurring and experimentally induced, a considerable number of 50 A filaments appear in the denser cytoplasmic cortex, but only in the walls of the furrow. These filaments are similar to those which have been demonstrated in a number of contractile cells. Accordingly, it is suggested that this fibrillar system may be actively involved in the development of the cleavage force.     

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography.
Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave.
Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.     

ON PROPAGATION OF CORTICLA FACTOR AND CYTOPLASMIC FACTOR PARTICIPATING IN CLEAVAGE FURROW FORMATION OF THE NEWT'S EGG*

From Cynops pyrrhogaster eggs just after the start of the first cleavage, a fragment of cortical layer with a small entire cleavage furrow was cut out. In the fragment, the cortex had already acquired susceptibility to and the subcortical cytoplasm had already accquired inducibility for furrow formation. The fragment was transplanted to the animal hemisphere of uncleaved fertilized eggs or eggs immediately after the onset of the first cleavage, from which a portion of the host cortex was removed. Observation was made on division of the graft, and on propagation of the cortical susceptibility and the cytoplasmic inducibility of the graft onto the host egg. The transplant divided succesively on the host egg in many cases, but the furrow of the graft never advanced to the surface of the host egg. Neither the cortical factor nor the cytoplasmic factor was transmitted across the graft to the recipient egg.     

THE EXTRACELLULAR RELEASE OF ECHINOCHROME

A study was made of the diffusion of the red pigment echinochrome from the eggs of the sea urchin, Arbacia punctulata, into sea water. Unfertilized eggs retained their pigment, over periods of hours. Outward diffusion of pigment from unfertilized eggs normally is entirely negligible, or does not occur at all. Enchancing the calcium or potassium content of the artificial sea water (while retaining isosmotic conditions) did not induce pigment release. Under anaerobic conditions, unfertilized eggs release pigment in small quantities. Fertilization alone brings about echinochrome release. Fertilized eggs invariably released pigment, whether in normal sea water, or sea water with increased calcium or potassium. This diffusion of the pigment began during the first cleavage, possibly soon after fertilization. The pigment release is not a consequence solely of the cell''s permeability to echinochrome (or chromoprotein, or other pigment combination) but is preceded by events leading to a release of echinochrome from the granules in which it is concentrated within the cell. These events may be initiated by activation or by anaerobiosis. The phenomenon was not due to cytolysis.     

A possible bio-assay for chromosome movement in isolated mitotic apparatus

We isolated mitotic apparatus (MA) from sea urchin zygotes using several methods. The isolated MA were injected into nucleated frog eggs or into enucleated frog eggs, and 24 h later we determined whether or not the eggs had undergone normal cleavage. Normal cleavage occurred in about 50% of the cases when MA isolated in glycerol-dimethyl sulphoxide were injected into either nucleated or enucleated frog eggs. Likewise for MA isolated initially in hexylene glycol and transferred immediately into glycerol-dimethyl sulphoxide. No cleavage occurred when MA isolated in hexylene glycol (and stored in hexylene glycol) were injected into frog eggs. We discuss two possible interpretations of the results. In one interpretation cleavage of the frog eggs is a bioassay, measuring the ability of the isolated MA to support chromosome movement. In the other interpretation the isolated MA contribute only nuclear material and cleavage initiation factors, and there is no chromosome movement in the isolated MA per se.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

RELATIONSHIPS BETWEEN DISTRIBUTION OF TUBULIN-PARACRYSTALS,GRANULES STAINING WITH NEUTRAL RED AND CLEAVAGE ACTIVITY IN SEA URCHIN EGG-FRAGMENTS1,2

Unfertilized eggs of Japanese sea urchins (Temnopleurus toreumaticus and Hemicentrotus pulcherrimus)were separated by centrifugation into two fractions (nucleated light and enucleated heavy fragments) or three fractions (nucleated light, enucleated middle, and enucleated heavy fragments). These fragments were stained with neutral red and then fertilized. Cleavage took place only in fragments containing cytoplasmic granules staining with neutral red: no cleavage occurred in fragment without these granules. When fragments of unfertilized eggs were incubated in a solution in sea water of 10^?4M vinblastine, a mitotic poison that specifically binds to tubulin, tubulin-paracrystals were found in all kinds of fragments, irrespective of whether they had stained granules and cleavage activity. These results suggest that lack of cleavage activity in the fragments is not due to the absence of polymerizable tubulin molecules in the cytoplasm, but rather to other factors, such as the absence of granules staining with neutral red. In other words, there is no relation between the distribution of these granules and polymerizable tubulin, but a close relation between the number of stainable granules and cleavage activity. Quantitative analysis of tubulin molecules in the egg fragments is necessary for confirmation of this idea.     

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.     

WAVE OF STIFFNESS PROPAGATING ALONG THE SURFACE OF THE NEWT EGG DURING CLEAVAGE

In the eggs of the newt, Cynops (Triturus) pyrrhogaster, change in stiffness of the cortex was measured in various regions at the time of the cleavage. Measurements were performed by Mitchison and Swann's cell elastimeter method with a modification, in which two fine pipettes were attached to the surface of one egg at the same time, in order to compare the rigidity of two regions. The stiffness of the cortex changed very little before the start of the first cleavage. However, just before the appearance of the first cleavage furrow, the stiffness increased rapidly at the animal pole region, which later returned to the former level. As the cleavage furrow progressed, a wave of high stiffness travelled meridionally as a belt along the surface from the animal pole region toward the vegetal region. At second cleavage, the cycle of change in stiffness was repeated.     

REFRACTIVE INDEX OF THE PROTOPLASM IN SEA URCHIN EGGS*

The distribution of the refractive index (RI) of the protoplasm in sea urchin eggs was determined from the optical path differences at various regions of the cell measured by interference microscopy assuming that the cell structure is symmetrical about the line passing through the center of the cell and that of the nucleus in unfertilized eggs and about the spindle axis in fertilized eggs during mitosis and cleavage. The RI of the cytoplasm in the unfertilized egg was uniform except for the cortical region, which had the RI higher than that of the underlying endoplasm. The RI of the cortex was generally higher than that of the underlying endoplasm, which did not appreciably change during mitosis and cleavage. The RI of the nucleus was lower than that of the cytoplasm. The RI of the mitotic apparatus was lower than that of the surrounding cytoplasm. The fertilization membrane had a thickness of about 0.6 μm in hydrated state and about 25 nm in dried state (mean values). The RI of the perivitelline space was about 0.00015 higher than that of seawater, equivalent to 0.08 g/100 ml of contents.     

Cleavage inhibition in marine eggs by puromycin and 6-dimethylaminopurine

The possibility is discussed that puromycin may not inhibit cleavage in marine eggs solely by inhibiting protein synthesis. It is suggested that part of the effect of puromycin is through 6-dimethylaminopurine (DMAP) (the purine component of puromycin) a potent cleavage inhibitor which appears to enhance protein synthesis in sea urchin eggs.     

Effects of DNP- and NaN3-pretreatment on cleavage of the sea urchin egg

Unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus were treated with DNP-sea water (M/500 - M/2,000) or with NaN₃-sea water (M/50 - M/800) for 15 – 90 minutes, and 2 hours later they were inseminated. Such a treatment induced acceleration of the first cleavage, and the eggs had a tendency to divide directly into 4 cells in the first cleavage cycle. In another series of experiments, unfertilized eggs were developed parthenogenetically by Loeb 's double-treatment after the above-mentioned treatment. It was found that these eggs divided earlier than the controls and that they developed into swimming blastulae in higher percentages. From the results described in the present and other papers it is suggested that DNP and NaN₃ have effects to activate eggs parthenogenetically and also to induce division of the centrioles, closely relating to the cleavage.     

Morphological criteria of egg quality in marine fishes: Activation and cleavage of eggs of <Emphasis Type="Italic">Zebrasoma scopas</Emphasis> (Acanthuridae)

Morphological changes of the eggs of Zebrasoma scopas obtained just after ovulation and after the storage of ovulated oocytes in the ovarian cavity of the female (in vivo) or in the external medium (in vitro) are described. The dynamics of cortical reaction is followed in noninseminated oocytes, as well as in inseminated oocytes of various quality. The average proportions of eggs exhibiting normal cleavage in the control series (comprised the eggs inseminated just after ovulation) and in the series stored in vivo for 2 and 4 h at 27°C are 83, 30, and 35%, respectively. The average proportions of eggs with normal cleavage in the control series and in the series stored in vitro for 2 and 4 h at 25°C are 88, 79, and 34%, respectively. The storage of oocytes in vitro at 7°C for 2 h leads to the abrupt decrease of their quality with the proportion of normally cleaved eggs reaching only 15%. The types of abnormal cleavage leading to subsequent degradation of cells of the blastodisc and total mortality of eggs are as follows: (1) mitotic cleavage of nuclei accompanied by incomplete cytotomy, (2) mitotic cleavage of nuclei without cytotomy, (3) a weak adhesion between cells, and (4) desynchronization of cell divisions. The methods for the assessment of egg quality just after ovulation and at initial developmental stages are suggested for marine teleost fishes. Published in Russian in Voprosy Ikhtiologii, 2008, Vol. 48, No. 4, pp. 537–552. The article was translated by the authors.