THE EFFECT OF PENICILLIN ON EGGS OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. Penicillin in the range of concentration from 250 U/ml. to approximately 2650 U/ml. inhibits the rate of cell division of the fertilized sea urchin egg from 0 to 100 per cent. 2. Penicillin in the same range of concentrations has no effect on the oxygen consumption of the unfertilized or the fertilized eggs. 3. Penicillin is bound by some component of the sea urchin egg in amounts sufficiently large to lower the initial concentration, this binding apparently not being related to the inhibitory action.     

A CYTOLOGICAL STUDY OF ARTIFICIAL PARTHENOGENESIS IN THE SEA URCHIN ARBACIA PUNCTULATA

Eggs of the sea urchin Arbacia punctulata were artificially activated with hypertonic seawater. The artificially activated eggs undergo the cortical reaction which is not distinguished by a wavelike progression as in the case of inseminated eggs. The cortical granules are released at random loci at the surface of the egg and result in spaces separated by large cytoplasmic projections. Unreacted cortical granules and ribosomes are found within the matrix comprising the large cytoplasmic projections. No "fertilization cone" is formed. The subsequent release of additional cortical granules results in the formation of a continuous perivitelline space, 15 min following activation. 85 min postactivation, an organization of annulate lamellae, endoplasmic reticulum of the smooth variety, and microtubules around a centriole is observed prior to nuclear division. Before the breakdown of the nuclear envelope a streak stage is formed. The streak is composed of a central core of annulate lamellae and is encompassed by endoplasmic reticulum and vesicular components. Condensation of chromatin is followed by the establishment of the mitotic apparatus. Centrioles were not found in the mature egg; however, they are present after activation prior to the first nuclear division, in the four-cell embryo, multicellular embryo, and at blastula. Artificially activated eggs have been observed to develop to the pluteus stage in more than 50% of the eggs treated.     

A CYTOLOGICAL STUDY OF THE CENTRIFUGED WHOLE, HALF, AND QUARTER EGGS OF THE SEA URCHIN ARBACIA PUNCTULATA

While the ooplasmic components of centrifuged eggs of Arbacia punctulata do not stratify in homogeneous layers, we have obtained the following strata beginning with the centripetal end: lipid droplets, pronucleus, clear zone, mitochondria, yolk, and pigment. Whereas mitochondria may be found mingled with yolk bodies, we have never observed lipid droplets nor pigment bodies among any of the other inclusions. The so-called clear zone contains a heterogeneous population of inclusions: annulate lamellae, heavy bodies, Golgi complexes, and rod-containing vacuoles. The peripheral cortical granules of immature (germinal vesicle stage) and of mature eggs are not dislodged from the cortical ooplasm with the centrifugal force utilized. When the eggs are treated with urethane, prior to centrifugation, the cortical granules of mature eggs abandon their peripheral position. Further centrifugation of the initially stratified eggs produces nucleated and nonnucleated halves and the centrifugation of the halves results in quarters. The cytology of the halves and quarters is discussed. The halves and quarters have been activated with either sperm or hypertonic sea water. With the exception of the nucleated halves, we were unable to obtain plutei larvae from the other fractions (red halves and quarters). We believe that the lack of development of the various fragments is a function of the balance of particular inclusions necessary for differentiation.     

THE EFFECTS OF NICOTINE ON FERTILIZATION IN THE SEA URCHIN, ARBACIA PUNCTULATA

The number of sperm incorporated into eggs made polyspermic with varying concentrations of nicotine (0.025–0.25%, v/v) appears to be directly related to the concentrations employed. The cortical response is morphologically equivalent to that observed in control preparations. Shortly after their incorporation all of the spermatozoa undergo structural events normally associated with the development of the male pronucleus in monospermic eggs. During the reorganization of the spermatozoa, sperm asters are formed. The number of male pronuclei that initially migrate to and encounter the female pronucleus is usually one to three. When pronuclei come into proximity to one another the surface of the female pronucleus proximal to the advancing male pronuclei flattens and becomes highly convoluted. Subsequently, the pronuclei contact each other and the outer and inner membranes of the pronuclear envelopes fuse, thereby producing the zygote nucleus. The male pronuclei remaining in the zygote after this initial series of pronuclear fusions continue to differentiate, i.e. they enlarge, form nucleolus-like bodies, and undergo further chromatin dispersion. In approximately 90% of the zygotes, all of the remaining male pronuclei progressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 min postinsemination. Mitosis and cleavage of the polyspermic zygote occurs later than in monospermic eggs.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

THE OSMOTIC PROPERTIES OF LIVING CELLS (EGGS OF ARBACIA PUNCTULATA)

We have attempted to answer the question: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight.     

INCREASED PERMEABILITY TO WATER OF AGING UNFERTILIZED EGGS (ARBACIA PUNCTULATA)

1. Unfertilized Arbacia eggs, subjected to 50, 60, or 80 per cent sea water, at the same temperature, pH, and duration, swelled progressively faster with age. Swelling increased 12.2 to 39.6 per cent. 2. Permeability to water was calculated by the method of Lucké and McCutcheon. For freshly shed eggs from nine different females, permeability varied from 0.078 to 0.124, or 59 per cent. This variation was not due to differences in age nor to physiologic condition but was correlated with size of eggs. 3. About twenty eggs were tested each age to 60 hours after shedding. The permeability rate increased with age in all nine experiments. 4. The total increase ranged from 30 to 69 per cent. 5. The increase was slow but constant or almost constant during early and intermediate ages. 6. The increase began at earliest ages. 7. An increase may not be demonstrable for any short interval but is readily demonstrable for longer intervals (6 or more hours), or, from the entire curve. 8. Permeability increased faster at late ages. For special factors during late ages see text. 9. Injury by experimental procedure was not induced until ca. the 24th hour. Injury by aging was not detectable during the first 6 to 9 hours. It increased progressively between 10 and 24 hours, and very rapidly thereafter. Permeability increased at approximately the same rate during early and intermediate ages. Permeability therefore was not correlated with degree of injury. 10. The troublesome factor in the permeability equation was initial size, or V_o. Several methods were used to obtain this V_o, viz. (a) extrapolations approximating known cyclical change in size, (b) extrapolations approximating control sizes, (c) constant or average of all V_o''s, (d) maximum V_o approximating control V_o''s. Recalculation with these different V_o''s gave approximately the same permeabilities. 11. The increase in permeability with age was substantially the same whether "approved," or other possible rates or average rates or range of rates be used as criteria of change. 12. When the data were recalculated by integrating the equation of Northrop, the increases in permeability with age were essentially the same as the increases calculated by the graphic method of Lucké.     

THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.     

A CYTOLOGICAL STUDY OF THE RELATION OF THE CORTICAL REACTION TO SUBSEQUENT EVENTS OF FERTILIZATION IN URETHANE-TREATED EGGS OF THE SEA URCHIN, ARBACIA PUNCTULATA

Eggs of the sea urchin, Arbacia punctulata, treated with 3% urethane for 30 sec followed by 0.3% urethane and inseminated are polyspermic and fail to undergo a typical cortical reaction. Upon insemination the vitelline layer of urethane-treated eggs either does not separate or is raised only a short distance from the oolemma. 1–6 min after insemination, almost all of the cortical granules remain intact and are dislodged from the plasmalemma. Later (6 min to the two-cell stage) some cortical granules are released randomly along the surface of the zygote. Not all zygotes show the same degree of cortical granule dehiscence; most of them experience little if any granule release whereas others demonstrate considerably more. The thickness of the hyaline layer appears to be directly related to the number of cortical granules released. Subsequent to pronuclear migration, several male pronuclei become associated with the female pronucleus. Later the male and female pronuclear envelopes contact and the outer and the inner laminae fuse, thereby forming the zygote nucleus. The male pronuclei remaining in the cytoplasm increase in size and progressively migrate to, and fuse with, the zygote nucleus. By 60 min some zygotes appear to contain only one large zygote nucleus which subsequently enters mitosis. Other zygotes possess a number of male pronuclei which remain unfused, and later these pronuclei along with the zygote nucleus undergo mitosis. There does not appear to be a direct relation between the number of cortical granules a zygote possesses and the above mentioned dichotomy.     

ON THE RATE OF OXYGEN CONSUMPTION BY FERTILIZED AND UNFERTILIZED EGGS : IV. CHAETOPTERHS AND ARBACIA PUNCTULATA

1. Unfertilized eggs of Chaetopterus consume about 2.4 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 2. In the 1st hour after fertilization, the fertilized eggs consume oxygen at about 53 or 54 per cent of this rate, which is about 1.3 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 3. For the first 6 hours after fertilization, at 21°C., the curve of the rate of oxygen consumption is slightly asymmetrically sigmoid. The prefertilization rate is regained between 4½ and 5 hours after fertilization. Soon after 6 hours, ciliary activity begins, and the rate of oxygen consumption rises rapidly. 4. The unfertilized eggs of Arbacia punctulata consume about 0.36–0.5 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. The absolute determination is difficult as these eggs are highly sensitive to shaking in the manometer vessels, and these difficulties are discussed. 5. The fertilized eggs of Arbacia punctulata consume oxygen at the rate of about 2.0 mm.³ O₂ per hour per 10 mm.³ 21°C. At 1 hour after fertilization the rate is already rising. 6. A comparison of the absolute rates of oxygen consumption, and the changes in rate at fertilization of these and a number of other eggs, together with a theoretical discussion, and a discussion of discrepancies in measurements on the eggs of Arbacia punctulata, is contained in the fifth paper of this series (21).     

FERTILIZATION AND THE TEMPERATURE COEFFICIENTS OF OXYGEN CONSUMPTION IN EGGS OF ARBACIA PUNCTULATA

The eggs of A. punctulata have a high temperature coefficient in the resting state: Q ₁₀ = 4.1. On fertilization and on cytolysis the temperature coefficient falls to less than half the resting value: Q ₁₀ = 1.8 and 1.9 respectively. The factor by which oxygen consumption increases on fertilization is a variable, its magnitude depending on temperature as well as on egg species. It is nearly ten times greater at 11°C. and only double at 29.9°C. By extrapolating to 32°C. there would be no increase on fertilization. Critical thermal increments common to many oxidations, 6,500, 10,800, and 12,500, have been found. The possible significance of these results is discussed in relation to the catalytic mechanisms and structural organization of the egg cell.     

MICROINJECTION OF COLCHICINE INTO SEA URCHIN EGGS

Inhibition of cleavage by colchicine was examined by microinjecting colchicine solution into one of the blastomeres of a sea urchin egg at the two-cell stage. Cleavage was inhibited if the microinjection was made before a critical point prior to the cleavage, whereas cleavage occurred in spite of the destruction of the mitotic apparatus if the microinjection was made after the critical point. The critical point was 10 min before the mid-stage of the cleavage in Clypeaster japonicus and 8 min before the mid-stage in Temnopleurus toreumaticus at 20 ± 1°C, corresponding to the beginning of anaphase. The threshold for the cleavage inhibition of colchicine was estimated to be 3 × 10⁻⁵ M to 3 × 10⁻⁶ M in final concentration in the cell.     

THE EFFECTS OF URETHANE AND CHLORAL HYDRATE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

The effects of a series of concentrations of the narcotics, ethyl carbamate and chloral hydrate, have been determined on the consumption of oxygen by fertilized and unfertilized eggs of the sea urchin Arbacia punctulata. In the fertilized eggs the effects of the two inhibitors on cell division were also examined. The following observations were made: 1. Assuming that the narcotic acts upon a single catalyst in the unfertilized egg the degree to which the consumption of oxygen is inhibited in this resting cell can be related to the narcotic concentration by an expression derived from the law of mass action. 2. To account for the relation between the concentration of the narcotic and its effect on respiration in the fertilized eggs, it is necessary to conclude that in them the narcotic acts on two parallel respiratory systems. The experimental data can be quantitatively predicted (1) if the reaction of the narcotic on the two systems is governed by the law of mass action and (2) if 40 per cent of the oxygen consumption is mediated by one system, the "activity" system, and the remainder by the other, the "resting" or "basal" system. 3. The mass law constants applying to the resting system in the fertilized egg are similar to those for the single system functioning in the unfertilized egg so that these two respiratory systems are probably identical. 4. The concentrations of the narcotics just sufficient to abolish cell division affect primarily the activity system, the existence of which was inferred from the respiratory experiments. It is concluded that normal cell division requires specifically the normal function of the activity system, that in fact the energy for cell division is made available through that system.     

MICROPAPILLAE IN SEA URCHIN EGGS*

Sea urchin eggs fixed in a glutaraldehyde-calcium chloride mixture have “micropapillae” with a dense content. If these structures are real, they are likely to be sites where a fusion with the spermatozoon can take place. It is possible however that they represent some kind of preparation artefact without a structural counterpart in the living state.     

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.     

THE BEHAVIOR OF THE NUCLEIC ACIDS DURING THE EARLY DEVELOPMENT OF THE SEA URCHIN EGG (ARBACIA)

The authors wish to correct an error in the paper "The behavior of the nucleic acids during the early development of the sea urchin egg (Arbacia)" (J. Gen. Physiol., 1947–48, 31, 203). Owing to an oversight, the figures for the amounts of various P fractions in a single Arbacia egg have been erroneously expressed in γ x 10^–3 units (Tables I and II, page 205; the last two lines of page 206). The figures should have been expressed in γ x 10^–5 units. Thus, the fertilized Arbacia egg contains an average of 20 γ x 10^–5 ribonucleic acid P and 0.7 to 1 γ x 10^–5 desoxyribonucleic acid P.     

FORCE EXERTED BY THE CLEAVAGE FURROW OF SEA URCHIN EGGS

A drop of ferrofluid injected into the center of a dividing sea urchin egg is deformed into the shape of an hourglass when the cleavage furrow advances. The force applied to the drop is determined from the deformation of the drop and the interfacial tension between the ferrofluid and the protoplasm. The interfacial tension is determined from the deformation of a spherical drop in the protoplasm when a magnetic field is applied, and the force applied to the drop, which is estimated from the deformation by magnetic field of a similar drop in 2 per cent aqueous solution of Triton X-100 and the interfacial tension between the ferrofluid and this solution.
The force applied to the drop in the dividing egg increases during an early stage of cleavage and decreases during a later stage. The force attained a maximum of 9 × 10⁻³ dyne in an egg of Temnopleurus toreumaticus which pinched the drop into two when it divided. Smaller maximum forces, 3.9 × 10⁻³ dyne in the eggs of Temno-pleurus toreumaticus and 2.0 × 10⁻³ dyne in the eggs of Clypeaster japonicus (mean values), were obtained when the furrowing was arrested by the drop. The magnitude of the maximum tension developed in the contractile element located in the furrow cortex is discussed.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VIII. THE DIPHOSPHOPYRIDINE NUCLEOTIDE (COZYMASE) CONTENT OF EGGS OF ARBACIA PUNCTULATA

1. The diphosphopyridine nucleotide content of Arbacia eggs has been measured manometrically and found to be approximately 250–500 micrograms per gm. wet weight of eggs, the value varying with individual egg samples and with the state of development of the eggs. Of the total diphosphopyridine nucleotide present, approximately 25–40 per cent is in an alkali-stable, presumably the dihydro, form. 2. Tests for triosephosphate and glutamic acid dehydrogenases carried out on Arbacia egg cytolysates were negative.