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.     

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.     

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.     

REINSEMINATION OF FERTILIZED SEA URCHIN (ARBACIA PUNCTULATA) EGGS

Embryos of Arbacia punctulata , treated to remove the fertilization membrane and hyaline layer, were mixed with sperm (reinseminated) at 20–30 min (streak stage) and at 70 min (2-cell stage) postinsemination. Sperm, incorporated into embryos reinseminated at the streak stage, metamorphosed into male pronuclei which subsequently migrated to and fused with the zygote nucleus. Although blastomeres were capable of incorporating sperm and forming fertilization cones, less than 10% of the 2-cell stage embryos reinseminated. Sperm, were found entrapped within an amorphous material along the surface of 2-cell stage embryos; many had failed to undergo an acrosome reaction. These results indicate that conditions necessary for incorporation and metamorphosis of sperm nuclei into male pronuclei are present in the embryo after the normal period of fertilization.     

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.     

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.     

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.     

OOCYTE DIFFERENTIATION IN THE SEA URCHIN, ARBACIA PUNCTULATA, WITH PARTICULAR REFERENCE TO THE ORIGIN OF CORTICAL GRANULES AND THEIR PARTICIPATION IN THE CORTICAL REACTION

This paper presents morphological evidence on the origin of cortical granules in the oocytes of Arbacia punctulata and other echinoderms. During oocyte differentiation, those Golgi complexes associated with the production of cortical granules are composed of numerous saccules with companion vesicles. Each element of the Golgi complex contains a rather dense homogeneous substance. The vesicular component of the Golgi complex is thought to be derived from the saccular member by a pinching-off process. The pinched-off vesicles are viewed as containers of the precursor(s) of the cortical granules. In time, they coalesce and form a mature cortical granule whose content is bounded by a unit membrane. Thus, it is asserted that the Golgi complex is involved in both the synthesis and concentration of precursors utilized in the construction of the cortical granule. Immediately after the egg is activated by the sperm the primary envelope becomes detached from the oolemma, thereby forming what we have called the activation calyx (see Discussion). Subsequent to the elaboration of the activation calyx, the contents of cortical granules are released (cortical reaction) into the perivitelline space. The discharge of the constituents of a cortical granule is accomplished by the union of its encompassing unit membrane, in several places, with the oolemma.     

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.     

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.     

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.     

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.     

MICROTUBULES IN THE FORMATION AND DEVELOPMENT OF THE PRIMARY MESENCHYME IN ARBACIA PUNCTULATA : I. The Distribution of Microtubules

Prior to gastrulation, the microtubules in the presumptive primary mesenchyme cells appear to diverge from points (satellites) in close association with the basal body of the cilium; from here most of the microtubules extend basally down the lateral margins of the cell. As these cells begin their migration into the blastocoel, they lose their cilia and adopt a spherical form. At the center of these newly formed mesenchyme cells is a centriole on which the microtubules directly converge and from which they radiate in all directions. Later these same cells develop slender pseudopodia containing large numbers of microtubules; the pseudopodia come into contact and fuse to form a "cable" of cytoplasm. Microtubules are now distributed parallel to the long axis of the cable and parallel to the stalks which connect the cell bodies of the mesenchyme cells to the cable. Microtubules are no longer connected to the centrioles in the cell bodies. On the basis of these observations we suggest that microtubules are a morphological expression of a framework which opeartes to shape cells. Since at each stage in the developmental sequence microtubules appear to originate (or insert) on different sites in the cytoplasm, the possibility is discussed that these sites may ultimately control the distribution of the microtubules and thus the developmental sequence of form changes.     

荒漠藻壳的精细结构与发育

以宁夏沙坡头不同时期围栏形成的固沙地为样点,将土壤学研究手段与土壤微生物学研究方法相结合,首次在微米层次揭示了荒漠藻壳中藻类植物的自然群落结构和空间分布规律,同时结合矿物物相分析,藻类生物量、土壤理化性质,从生物学、土壤学、矿物学交叉结合的角度深入地揭示了荒漠藻壳的结构和发育,为荒漠拓殖生物群落的发育和人工调控荒漠藻壳固沙培肥技术的应用提供了最直接的依据。另外研究中所采用的多学科交叉结合的实验手段     

OCCURRENCE OF MUCOPOLYSACCHARIDES IN THE EARLY DEVELOPMENT OF THE SEA URCHIN EMBRYO AND ITS ROLE IN GASTRULATION

The occurrence of acid mucopolysaccharides in the early development of the sea urchin embryo was studied by histochemical stainings as well as by autoradiographic methods. By histochemical methods acid niucopolysacchdride was demonstrated at the vegetal region in the early stage of gastrulation as a globular structure. Experiments with ³⁵S-labeled sulfate which was incorporated into acid mucopolysaccharides confirmed the result obtained by histochemical observation. It was revealed that sulfate polysaccharide in the vegetal region moved toward the blastocoel in parallel with the shedding of the primary mesenchyme cells. When the incorporation of sulfate into the acid mucopolysaccharides was inhibited by selenate, the primitive gut development was remarkably repressed. The substance seems to be indispensable for smooth cell movements essential for the gastrulation of sea urchin embryo.     

THE CENTRAL ROLE OF THE GENITAL DUCT IN THE DEVELOPMENT AND REGENERATION OF THE GENITAL ORGANS IN THE SEA URCHIN

Formation and regeneration of the genital papilla in the sea urchin, Hemicentrotus pulcherrimus were studied to find out what factor(s) control expression of the secondary sexual characters in the papilla. Surgical removal of various regions of the genital organ and the blood vessels was performed separately or in various combinations. The proximal part of the genital duct or its rudiment (D2 region) plays an essential role in the formation or regeneration of the papilla, and in the establishment of its sexual characters as well, i.e., if the D2 region is removed, papilla formation or regeneration is completely suppressed; when the D2 region is left uninjured, the papilla develops and displays the sexual characters even in the absence of the gonad, the distal part of the duct, a blood supply to the D2 region or all three of them combined. It can be concluded that the proximal part of the genital duct carries the factor(s) for determination and expression of the secondary sexual characters in the genital papilla. Discussion has been made about what factor(s) can be considered to reside within the cells of the D2 region of the duct.     

MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13).     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.