Ultrastructural study of the mature egg of Tethya citrina sarà and melone (porifera,demospongiae)

Tethya citrina is an oviparous demosponge in which eggs are distributed in clumps within the choanosome. The cytoplasm of the mature egg presents a peripheral cortex consisting of a slightly granular layer sandwiched between two densely granular, vesiculated ones. The cortex probably has a specialized, trophic function. Mesohyl bacteria are phagocyted at the egg surface, included in vacuoles, and transferred across the cortical sheath toward the inner cytoplasm. The region of the egg extending between the cortex and the nucleus shows a lacunary system mostly developed beneath the cortical envelope. The noncortical cytoplasm also contains lipid droplets, dense rodlike bodies, and phagosomelike granules. Most of the latter are probably autophagosomes, forming lacunae and supporting autosynthetic vitellogenesis. Rodlike inclusions are probably proteinaceous; they likely originate within the phagosomes and represent the actual yolk material.     

Cytochemistry of yolk elements in the Amphioxus egg

Summary The Amphioxus egg develops compound yolk and lipid yolk, besides the cortical vacuoles described previously. The compound yolk elements consisting of carbohydrates, proteins, lipoproteins, and triglycerides originate within the ooplasmic masses that are constituted by the yolk nucleus substance and mitochondria. The lipid yolk elements, which are poorly developed, stain for phospholipids; the exact mode of their formation could not be determined. The behaviour and cytochemistry of organelles (yolk nucleus and mitochondria) have also been described.     

Histochemical nature of cortical granules in the human egg

Summary The distribution and histochemical nature of cortical granules have been studied in the human egg. They are distributed as small granules adjacent to the plasma membrane of growing oocytes, and consist of carbohydrates and possibly some protein. The cortical granules of the human egg have been compared and contrasted to those in Amphioxus, fishes, and amphibians.     

SECONDARY PULVINUS OF ROBINIA PSEUDOACACIA (LEGUMINOSAE): STRUCTURAL AND ULTRASTRUCTURAL FEATURES

The structure of the secondary pulvinus of Robinia pseudoacacia has been examined together with ultrastructural features of motor cells both in open and closed pulvini, to identify ultrastructural changes associated with leaflet movement. Pulvini have a central vascular core bordered by thick-walled collenchyma cells, which in turn are surrounded by several layers of cortical parenchyma cells. Cortical motor cells exhibit ultrastructural features similar to those reported in homologous cells of other pulvini. The vacuolar compartment contains two kinds of vacuoles: nontannin vacuoles, which change both in number and size during leaflet movement, and tannin vacuoles, which may act as an ion reservoir. No differences in wall thickness were found between flexor and extensor motor cells. Thick walls of collenchyma cells show numerous pits with plasmodesmata through which the phloem parenchyma cells and the inner cortical motor cells are connected. Tannin vacuoles and calcium oxalate crystals are common inclusions of phloem parenchyma cells. The tissue arrangement and the occurrence of pits with plasmodesmata in the central cylinder cells provide evidence of symplastic continuity through the central cylinder between the extensor and flexor regions of the motor organs. The greater amplitude of Robinia leaflet movements may be related to the extension of motor regions, the scarcity of lignification in the central vascular core, and the thin flexor walls.     

Ultrastructure of the cortical cell of the interrenal gland of the American bullfrog (Rana catesbeiana)

Summary The cortical cell of the interrenal gland of the American bullfrog, Rana catesbeiana, was examined in the electron microscope. These cells occur in small groups and cords and are quite irregular in shape. The cortical cell is reminiscent of adrenal cortical cells from other vertebrates. Liposomes are variable in size and density. Smooth endoplasmic reticulum is scant in amount and predominantly of the fine tubular type. Mitochondria have vesicular cristae, a dense matrix, and occasionally have blebs, vacuoles, and myelin-like whorls at their surfaces. Intimate morphological relationships are found among the Golgi apparatus, lysosomes, and liposomes, and among Golgi vacuoles, mitochondria, and liposomes. In addition microfibrils are a prominent feature of the cortical cell. The biochemical events of steroidogenesis in amphibia and other vertebrates are discussed in relationship to the organellar interrelations found in the bullfrog interrenal cortical cells. Based on the available chemical and morphological information a scheme is proposed of movement of the steroidal intermediates through the cell that tentatively identifies the localizations of the various enzyme systems involved in corticosteroidogenesis from acetate to corticosterone and aldosterone.Supported in part by N.I.H. Grant RR 06138. Health Sciences Advancement Award.     

X-ray microanalysis of ion distribution within root cortical cells of the halophyte Suaeda maritima (L.) Dum.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m^-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m^-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m^-3 of analysed volume.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

Ooplasmic segregation in theTubifex egg: Mode of pole plasm accumulation and possible involvement of microfilaments

Summary Ooplasmic segregation, i.e. the accumulation of pole plasm in theTubifex egg, consists of two steps: (1) Cytoplasm devoid of yolk granules and lipid droplets migrates toward the egg periphery and forms a continuous subcortical layer around the whole egg; (2) the subcortical cytoplasm moves along the surface toward the animal pole in the animal hemisphere and toward the vegetal pole in the vegetal hemisphere, and finally accumulates at both poles of the egg to form the animal and vegetal pole plasms. Whereas the subcortical layer increases in volume during the first step, it decreases during the second step. This is ascribed to the compact rearrangement in the subcortical layer of membraneous organelles such as endoplasmic reticulum and mitochondria. The number of membraneous organelles associated with the cortical layer increases during the second step. Electron microscopy reveals the presence of microfilaments not only in the cortical layer but also in the subcortical layer. Subcortical microfilaments link membraneous organelles to form networks; some are associated with bundles of cortical microfilaments. The thickness of the cortical layer differs regionally. The pattern of this difference does not change during the second step. On the other hand, the subcortical cytoplasm moves ahead of the stationary cortical layer. The accumulation of pole plasm is blocked by cytochalasin B but not by colchicine. The first step of this process is less sensitive to cytochalasin B than the second step, suggesting that these two steps are controlled by differnt mechanisms. The mechanical aspects of ooplasmic segregation in theTubifex egg are discussed in the light of the present observations.     

An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio

Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m² and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).     

Cortical granule exocytosis is coupled with membrane retrieval in the egg of Brachydanio

Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.     

Histochemical observations on the cortical granules in the eggs of Indian lizards

Summary The development, distribution and histochemical nature of cortical granules have been investigated in growing eggs of two species of Indian lizards (Hemidactylus flaviviridis Rüppel and Uromastix hardwickii). Numerous cortical granules develop in the peripheral ooplasm of growing oocytes and are finally arranged in the cortical cytoplasm of the egg. They consist of a carbohydrate-protein complex; most of the carbohydrate component is an acid mucopolysaccharide. The cortical granules in the eggs of lizards have been compared and contrasted to those in Amphioxus, fishes, amphibians, and mammals.     

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 FINE STRUCTURE OF OOGENESIS IN MARCHANTIA POLYMORPHA

Archegonium development, beginning with the archegonial initial and culminating in the mature egg, was studied with the electron microscope. The ultrastructural features of the beginning stages in development of the archegonium are relatively similar to one another. Plasmodesmata occur between all adjacent cells at this time. After the secondary central cell is formed these protoplasmic connections are lost, and both axial and parietal cell lineages begin to show signs of ultrastructural differentiation. The mature egg is characterized by cytoplasm rich in ribosomes and larger organelles. Mitochondria and simplified plastids commonly display a juxtaposed association. As far as could be ascertained the numerous plastids and mitochondria in the egg of Marchantia arise through division of preexisting organelles and are not formed anew from evaginations of the nucleus. Blebbing of the nucleus produces polymorphic organelles which appear to be pinched off into the cytoplasm. The mature egg also contains vacuoles and lipid bodies toward its periphery, while dictyosomes and extensive endoplasmic reticulum occur throughout. The space between the wall cells and the mature egg appears to contain an amorphous substance. No extra membrane was observed around the mature egg.     

Fertilization in Oikopleura dioica (Tunicata,Appendicularia): Acrosome reaction,cortical reaction and sperm-egg fusion

Summary Fine structural changes in the egg and sperm are described during gamete interaction in Oikopleura dioica, an appendicularian tunicate. The unfertilized egg has a vitelline layer 80 nm thick and a perivitelline space about 5 m wide. In the peripheral cytoplasm are a few cortical granules 0.6×0.7 m in diameter and areas rich in parallel cisternae of rough endoplasmic reticulum alternating with areas rich in long mitochondria. In the deeper cytoplasm the predominant organelles are multivesicular bodies. From 25 s to 60 s after insemination, the egg transiently elongates, although with no obvious cytoplasmic rearrangement, and the egg surface becomes bumpy. During this interval sperm enter the egg, and the cortical granules undergo exocytosis. After expulsion into the perivitelline space, the cortical granule contents do not appear to change their shape or blend with the vitelline layer, which neither elevates further nor loses its ability to bind sperm. On encountering the egg, the sperm undergoes an acrosome reaction involving exocytosis of the acrosome and production of an acrosomal tubule. The acrosomal contents bind the sperm to the vitelline layer, and the posterior portion of the acrosomal membrane and the anterior portion of the nuclear envelope evaginate together to form an acrosomal tubule, which fuses with the egg plasma membrane to form a fertilization cone. By 45 s after insemination, the sperm nucleus, centriole, mitochondrion and at least the anterior portion of the axoneme are within the fertilization cone. By 60 s sperm entry is complete. In having eggs with a cortical reaction and sperm with an acrosome reaction, O. dioica resembles echinoderms and enteropneusts and differs markedly from ascidian tunicates, which lack both these features. The relatively unmodified pattern of gamete interaction in O. dioica in comparison with the highly modified pattern in ascidians is difficult to reconcile with the neoteny theory that appendicularians have evolved via ascidian ancestors. The present results are more consistent with the idea that an appendicularian-like ancestor gave rise to ascidians.     

ANIMAL/VEGETAL DIFFERENCES IN CORTICAL GRANULE EXOCYTOSIS DURING ACTIVATION OF THE FROG EGG*

One of the more striking morphological events during egg activation is exocytosis of the cortical granules. In the frog egg, the wave of cortical granule exocytosis takes about 100 sec to traverse the animal half, and travels slower in the vegetal half. We examined cortical granule exoctyosis during activation with respect to this animal/vegetal difference. In eggs which were acquiring the ability to be activated (recovering from CO₂-intoxication or undergoing meiotic maturation), animal half cortical granules became capable of responding to activating stimuli prior to vegetal half ones. Since Ca²⁺ is involved in exocytosis, we examined the effect of Ca²⁺ on cortical granule breakdown in vitro. There was no difference in sensitivity to Ca²⁺ of cortical granules from immature vs. mature eggs, but animal half cortical granules were more sensistive to Ca²⁺ than vegetal half ones. Finally, we found that prick-activation of eggs at the vegetal pole was frequently unsuccessful but would occur when external Ca²⁺ was raised. These experiments show that there are regional differences in the frog egg with respect to cortical granule responsiveness, and they suggest that the differences are due to Ca²⁺ sensitivity.     

Cortical Granules of Sea Urchin Eggs do not Undergo Exocytosis at the Site of Sperm-egg Fusion*

Microscopic observations of sea urchin egg fertilization (phase contrast, Nomarski and transmission electron microscope) reveal that the cortical granules in the area of sperm egg-fusion do not undergo exocytosis. These intact granules remain associated with the sperm, moving into the egg cytoplasm with the entering sperm. This sperm-cortical granule association occurs before the sperm centriole affects microtubule organization and the sperm-cortical granule association is not affected by cytochalasin D or griseofulvin. We discuss the possibility that a reorganization of the egg cytoplasm ensues from the sperm-egg interaction at the site of sperm-egg fusion. Other possibilities are that the retention of cortical granules is not related to egg reorganization, but is necessary for successful sperm incorporation or reflects an unrelated component of the activation process.     

Local alteration of cortical actin in Xenopus eggs by the fertilizing sperm

Rhodamine phalloidin (Rph) staining was used to examine the microfilament organization of the Xenopus laevis egg cortex during the early stages of fertilization. Unactivated eggs possessed a cytochalasin B (CR)-insensitive Rph-stained matrix that was reorganized upon egg activation and diminished in the presence of CB. Xenopus laevis sperm caused a temporary local increase in Rph staining on the Xenopus cortex. In CB-treated eggs, the local increases of cortical Rph staining later changed to a Rph-free area. These temporary local increases of cortical Rph staining were also observed when Notophthalmus viridescens sperm fertilized Xenopus and Rana pipiens eggs, and were followed by the appearance of concentric rings of stained and unstained areas. Our data suggest that Xenopus and Notophthalmus sperm have activities that can both organize and disrupt the cortical filamentous actin of the Xenopus egg. © 1993 Wiley-Liss, Inc.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

Subcellular localization of Cd in the root cells of<Emphasis Type="Italic">Allium sativum</Emphasis> by electron energy loss spectroscopy

The ultrastructural investigation of the root cells ofAllium sativum L. exposed to three different concentrations of Cd (100 (AM, 1 μM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes — high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.     

Surface area change at fertilization: Resorption of the mosaic membrane

Eggs of Strongylocentrotus purpuratus (sea urchin) have a surface area of 41,000 μm² before fertilization as determined by quantitative transmission and scanning electron microscopy. Within a minute after fertilization 18,000 cortical vesicles contribute an additional 57,000 μm² to form a mosaic membrane with the original plasma membrane. However, by 16 min after fertilization the total area of the egg is only 45,000 μm², indicating a rapid resorption of surface. Calculations of surface area depend in large part upon the numbers and dimensions of microvilli, after careful compensations are made for specimen shrinkage. The 134,000 microvilli per egg are 0.35 μm long before fertilization. They elongate to 1.0 μm in the first few minutes and then soon shorten to 0.5 μm. Even at their longest, microvilli do not accommodate all of the surface area of cortical vesicle membrane. The merger of cortical vesicle membranes and the plasma membrane was demonstrated many years ago and is not in doubt; however, this study indicates that the resulting mosaic membrane is not a long-lived, simple arithmetic combination of its components. Rather, the mosaic membrane undergoes a rapid and dynamic shrinkage by a mechanism which is not apparent on the basis of egg topography alone. The absolute values of egg surface area and dynamic changes in the surface are discussed in relation to physiological events accompanying fertilization.