Oocyte-follicle cell differentiation in two species of amphineurans (Mollusca), Mopalia mucosa and Chaetopleura apiculata

Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs. As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies. In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.     

The structure and composition of aleurone grains in the barley aleurone layer

Summary Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.     

Cytochemical studies of developmental stages during oogenesis in Culex pipiens molestus (Forsk?l). I. Lipids and carbohydrates

Lipids and carbohydrates were studied in the polytrophic ovaries of Culex pipiens molestus during oogenesis. The cytoplasm of both the oocyte and the nurse cells contains lipid structures at all stages of development--granules in the early stages and spheres in the later stages. Intranuclear lipid bodies can be demonstrated in the oocyte and in the nurse cells. After leaving the nucleus, lipids are deposited in the peripheral cytoplasm. From the third to the seventh adult phase, lipid granules are concentrated in the area of the nurse cell and oocyte junction, indicating that lipids originate in the nurse cells and are transported from these to the oocyte. The follicular epithelial cells provide the oocyte with lipid material for fatty yolk synthesis and formation of the egg envelopes. Lipids are distributed similarly to the Golgi apparatus, indicating that there is a relationship between this organelle and fat formation. In the early stages, the cytoplasm of the oocyte, the nurse cells and the follicular epithelium contains glycogen granules. In the later stages these cells also contain mucopolysaccharides. The mucopolysaccharide yolk spheres are enclosed in vacuoles, while the chorion is composed of acid mucopolysaccharides. The follicular epithelium and vitelline membrane are of a mucopolysaccharide nature. A topographical relationship exists between the Golgi apparatus and the glycogen granules, indicating that this organelle also plays a role in glycogen synthesis.     

Cytochemical studies on vitellogenesis in Aspongopus obscurus (Hemiptera: Pentatomidae)

By employing cytochemical techniques it is possible to conclude that the oöcytes of Aspongopus obscurus develop fatty and compound yolks. The fatty yolk originates in close proximity to the Golgi bodies and is composed of neutral fat. Based on the source of origin, the compound yolk has been grouped into two categories: CY₁ and CY₂. While the CY₁ develops de novo in the central oöplasm, the CY₂ originates from precursors infiltrating into the oöcyte through the follicular epithelium. Both CY₁ and CY₂ are composed of carbohydrate (1:2 glycol group and glycogen), protein (tyrosine, histidine, NH₂, SH and SS groups) and RNA.     

Crystalline bodies in cervicovaginal smears. A cytochemical and immunochemical study

Of 35,000 consecutive cervicovaginal smears examined, 111 were found to contain crystalline bodies with a radiate structure. Ninety-seven of these 111 smears had been obtained from pregnant women. The crystalline bodies were generally found in the cervical mucus portion of the smears and were evoking a considerable inflammatory reaction around them. Histochemical and immunochemical stains indicated that they are primarily composed of protein-carbohydrate complexes of a nonimmune type. In an attempt to determine the site of formation of the crystalline bodies during pregnancy, cervical biopsies available in six of the above cases were examined for crystalline bodies, with positive results in one nonpregnant case. Furthermore, 500 endometria from cases of interrupted pregnancies were examined for the presence of such structures, with negative results. It appears that crystalline bodies in smears from pregnant women are formed from products of degenerated inflammatory and other exfoliated cells within stagnating cervical secretions and primarily within dilated cervical glands. In this respect, they are similar or identical to the pseudoactinomycotic radiate granules or pseudosulfur granules reported in histologic sections of the uterus and other regions of the body. Further studies are needed to conclusively determine the site and mechanisms of formation of these structures.     

Structure of Cyst Wall Precursors and Kinetics of Their Appearance During the Encystment of Laurentiella acuminata (Hypotrichida,Oxytrichidae)1

The encystment of Laurenliella acuminata was divided into five stages: stage A (precystic semitransparent cell with dark-globules), stage B (precystic transparent cell), stage C (precystic pigmented cell), stage D (spherical shape without cyst wall) and stage E (young resting cyst), on the basis of observations of changes in morphology and pigmentation during encystment. The duration of these stages was also established. Observations by electron microscopy confirmed that the cyst wall, composed of four layers, is derived from different kinds of precursors which are synthesized “de novo.” The ectocyst precursors are composed of stacks of between 5 and 12 small thin plates or discs; these stacks are about 0.9 μm in length and 0.06 μm in height. The mesocyst precursors are fibrillar bodies of variable shapes, about 2.4 μm in maximum length and 0.12–0.16 μm in diameter. These precursors appear in the cytoplasm of the precystic cell during the first precystic stage (stage A). The endocyst precursors are rounded bodies surrounded by a fine membrane, and their contents appeared similar to the endocyst. The granular layer precursors are spherical bodies about 0.1–0.2 μm in diameter, surrounded by a double membrane presenting ribosomes adhering to its outer membrane. Both endocyst and granular layer precursors are observed in the precystic cytoplasm from stage B. On the basis of ultrastructural studies, a formation and growth model of the cyst wall of the hypotrichous ciliate Laurentiella acuminata is proposed.     

Infrastructure and cytochemistry of the midgut of Petrobius maritimus Leach, correlated with its excretory and digestive function

The midgut of Petrobius maritimus Leach is composed of several cell types: columnar, goblet and two types of light cells. These cells originate in common regenerative nests, the columnar and goblets cells arise at the periphery and the light cells arise from the axial portion of the nests. Columnar cells are seen to secrete digestive enzymes, absorb products of digestion, store lipid and may also be involved in excretion since they contain spherical-shaped, cristalloid bodies containing salt deposits. These bodies are seen to be formed in the RER in association with tile Golgi complex. There is some evidence that columnar cells undergo degenerative change. Goblet cells contain proteinaceous granules formed from the Golgi complex. At last, one of these two types of light cells may be comparable to endocrine cells of the vertebrate digestive tract.     

Electron microscope studies on developing oocytes of a coelenterate medusa with special reference to vitellogenesis

In a hydrozoan jellyfish, the female gonad is differentiated from a specialized region of the epidermis near the manubrium. Changes in the oocytes during growth and vitellogenesis are described as observed with electron microscopic and cytochemical techniques. Three major types of yolk are formed; these include lipid, glycogen, and membrane-bound granules consisting of both protein and carbohydrate. The latter first appear evident within vesicular and cisternal elements of the numerous Golgi complexes. The orientation and structural variations noted between the endoplasmic reticulum and forming face of the Golgi complexes suggest that the protein component of the yolk granules may be transferred from the cisternae of the endoplasmic reticulum to the Golgi complex where it is joined to carbohydrate perhaps synthesized by the Golgi complexes. Stages in the release of the precursor yolk material sequestered in cisternal elements of the Golgi complexes are illustrated. The presence of coated and uncoated vesicles in the Golgi regions and their possible role in intracellular transport are described and discussed. The presence and possible method of morphogenesis of vesiculate yolk bodies are also described. What appear to represent invaginations of the oolemma extend into the ooplasm and display a special orientation with respect to lamellae of the rough-surfaced endoplasmic reticulum. Intraooplasmic synthesis appears to constitute the major pathway for protein-carbohydrate yolk deposition.     

Cytochemical and fine structural analysis of oogenesis in the gastropod, Ilyanassa obsoleta

An analysis of differentiating oocytes of the gastropod, Ilyanassa obsoleta, has been made by techniques of light and electron microscopy. Early previtellogenic oocytes are limited by a smooth surfaced oolemma and are associated with each other by maculae adhaerentes. Previtellogenic oocytes are also distinguished by a large nucleus containing randomly dispersed aggregates of chromatin. Within the ooplasm are Golgi complexes, mitochondria and a few cisternae of the rough endoplasmic reticulum. When vitellogenesis begins, the oolemma becomes morphologically specialized by the formation of microvilli. One also notices an increase in the number of organelles and inclusions such as lipid droplets. During vitellogenesis there is a dilation of the saccules of the Golgi complexes and cisternae of the endoplasmic reticulum. Associated with the Golgi complexes are small protein-carbohydrate yolk precursors encompassed by a membrane. These increase in size by fusing with each other. The “mature” yolk body is a membrane-bounded structure with a central striated core and a granular periphery. At maturity a major portion of the ooplasmic constituents such as as mitochondria and lipid droplets occupy the animal region while the bulk of the population of yolk bodies are situated in the vegetal hemisphere. The follicle cells incompletely encompass the developing oocyte. In addition to the regularly occurring organelles, follicle cells are characterized by the presence of large quantities of rough endoplasmic reticulum and Golgi complexes whose saccules are filled with a dense substance. Associated with the Golgi saccules are secretory droplets of varied size. Amongst the differentiating oocytes and follicle cells are Leydig cells. These cells are characterized by a large vacuole containing glycogen. A possible function for the follicle and Leydig cells is discussed.     

A study on the structural protein of the vitreous body (vitrosin)

A method is described for the isolation of a structural protein of the vitreous body, which has been named vitrosin. The analyses show that vitrosin is a viscous, thixotropic, fibrous protein. Electron micrographs reveal that vitrosin particles are long fibrils, averaging 250 A in width. The isoelectric point was found to be around pH 5.5 and the shrinkage temperature 60°C. Vitrosin is composed of a protein-carbohydrate complex. It contains cystine and the aromatic amino acids in low quantities. Hexosamine could not be detected in the complex.     

Restoration of glycogenesis in hepatocytes from starved rats.

Hepatocytes isolated from the liver of rats starved for two days synthesized glycogen only when incubated in the presence of both glucose and glucogenic precursors (combinations of alanine, glycerol, pyruvate, lactate or fructose). Unlabeled glucogenic precursors facilitated the incorporation of [U-14C]glucose into glycogen. Unlabeled glucose likewise greatly enhanced glycogen synthesis from isotopically labeled lactate and other glucogenic precursors.In those systems which contained no added endocrines glucose dampened glycogen phosphorylase activity in a cAMP-independent fashion. Fructose is unable to mimic the effects of glucose on glycogen deposition and on glycogen phosphorylase activity.     

Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes.

Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.     

Sex-specific regionality of liver metabolism during starvation; with special reference to the heterogeneity of the lobular periphery

Staining procedures for glucose-6-phosphatase and 3-hydroxybutyrate dehydrogenase activity and for glycogen were used to investigate adaptive changes in the regionality of hepatic gluconeogenesis and ketogenesis in fasting male and female rats. A reciprocal distribution of gluconeogenic and ketogenic capacities was found in both sexes, but male and female animals were different with respect to: a) the time necessary for full induction of glucose-6-phosphatase activity (24 h in females, 48 h in males); b) the overall activity of 3-hydroxybutyrate dehydrogenase; and c) glycogen content. The activity of the latter enzyme and the glycogen content did increase with time of starvation, but at all times, were higher in males, than in females. Results, thus, indicate that the extent to which ketone bodies replace glucose as major fuel for the brain is larger in males than in females. This may explain the delayed induction of glucose-6-phosphatase activity and the higher glycogen content in the male during starvation. Distributions of enzyme activities and of glycogen, furthermore, revealed the heterogeneity of the lobular periphery, i.e. functional differences among sinusoids dependent upon whether they originate from the portal tract or the vascular septum, and thus confirm the lobular concept proposed by Matsumoto et al. (1979).     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during cell differentiation

1. The variation in cellular glycogen content of differentiating cells derived from myxamoebae that initially contained a wide range of glycogen contents (0.047-5.56mg of glycogen/10(8) myxamoebae) has been studied. 2. Myxamoebae that initially contained 0.047-3.62mg of glycogen/10(8) myxamoebae all gave rise to fruiting bodies that contained similar amounts of glycogen (0.06-0.11mg of glycogen/10(8) cells) but myxamoebae that initially contained 5.56mg of glycogen formed fruiting bodies containing 0.5mg of glycogen/10(8) cells. 3. Despite the high net rate of glycogen disappearance (during cell differentiation) from cells that contained more than 2mg of glycogen/10(8) cells initially, there were still significant variations in the rate of glycogen synthesis. The rate of glycogen synthesis reached a peak at the aggregation stage. 4. Evidence is presented showing that the rate of this synthesis of glycogen is controlled by factors other than the intracellular concentration of glycogen synthetase. 5. Our results are discussed in the context of the theory that the rates of glycogen synthesis and degradation act as a control mechanism for cell differentiation. 6. Criteria are discussed for deciding whether a biochemical event is causally or secondarily related to morphogenesis.     

Glycogen synthesis via the indirect gluconeogenic pathway in the periportal and via the direct glucose utilizing pathway in the perivenous zone of perfused rat liver

The isolated liver from 24 h fasted rats was perfused in a non-recirculating manner in the ortho- and retrograde direction with erythrocyte-containing (20% v/v) media to provide adequate oxygenation of the liver. Glucose and/or gluconeogenic precursors were added as substrates. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the sole exogenous substrate glycogen was deposited in the perivenous area, with gluconeogenic precursors it was formed in the periportal zone during ortho- and retrograde flow. When glucose and gluconeogenic compounds were offered together, glycogen was deposited in both zones. The results corroborate the model of metabolic zonation predicting that periportal glycogen is synthesized indirectly from gluconeogenic precursors while perivenous glycogen is formed directly from glucose.     

Glycogen synthesis via the indirect gluconeogenic pathway in the periportal and via the direct glucose utilizing pathway in the perivenous zone of perfused rat liver

Summary The isolated liver from 24 h fasted rats was perfused in a non-recirculating manner in the ortho-and retrograde direction with erythrocyte-containing (20% v/v) media to provide adequate oxygenation of the liver. Glucose and/or gluconeogenic precursors were added as substrates. Glycogen formation was determined biochemically and demonstrated histochemically. With glucose as the sole exogenous substrate glycogen was deposited in the perivenous area, with gluconeogenic precursors it was formed in the periportal zone during ortho-and retrograde flow. When glucose and gluconeogenic compounds were offered togethen, glycogen was deposited in both zones. The results cortoborate the model of metabolic zonation predicting that periportal glycogen is synthesized indirectly from gluconeogenic precursors while perivenous glycogen is formed directly from glucose.     

Electron microscope studies of Fasciola hepatica. XI. Autophagy and parenchymal cell function

An electron microscope study of the function of parenchymal cells in Fasciola hepatica in relation to glycogen storage and metabolism was undertaken.Although no evidence of a relationship between morphological changes in parenchymal cells and glycogen was apparent, a correlation between glycogen depletion and autophagy was observed. The autophagic process started with the synthesis and “budding off” of membranes by mitochondria to form small vesicles (M bodies), either of a simple type (limited by a single membrane) or a complex type (limited by two or more membranes). These M bodies fused to form narrow, smooth cisternae (SCM), which wrapped around areas of cytoplasm containing glycogen; this process gave rise to β bodies. The β bodies (autophagosomes) were at first irregular in shape and limited by two or more membranes separated by a space of varied width. Older β bodies became more smooth and oval in outline and were limited by a single membrane due to membrane fusion. Primary lysosomes synthesized by the GER-GA system united with the late β bodies and formed secondary lysosomes (autolysosomes). Following the addition of these lysosomal hydrolases, the glycogen content of the autolysosomes was reduced and eventually disappeared. This resulted in the develpment of residual bodies containing unhydrolysed material in the form of dense granules, tubules, and myelin figures in a matrix of varied density.It was concluded that at least some glycogen in the parenchymal cells of F. hepatica is mobilized by autophagy. All the morphological structures observed in the experimental material were also present in controls, although in fewer numbers, and it is believed that autophagy is a normal process in this fluke.     

Surfactant lipid synthesis and lamellar body formation in glycogen-laden type II cells

Pulmonary surfactant is a lipoprotein complex that functions to reduce surface tension at the air liquid interface in the alveolus of the mature lung. In late gestation glycogen-laden type II cells shift their metabolic program toward the synthesis of surfactant, of which phosphatidylcholine (PC) is by far the most abundant lipid. To investigate the cellular site of surfactant PC synthesis in these cells we determined the subcellular localization of two key enzymes for PC biosynthesis, fatty acid synthase (FAS) and CTP:phosphocholine cytidylyltransferase-alpha (CCT-alpha), and compared their localization with that of surfactant storage organelles, the lamellar bodies (LBs), and surfactant proteins (SPs) in fetal mouse lung. Ultrastructural analysis showed that immature and mature LBs were present within the glycogen pools of fetal type II cells. Multivesicular bodies were noted only in the cytoplasm. Immunogold electron microscopy (EM) revealed that the glycogen pools were the prominent cellular sites for FAS and CCT-alpha. Energy-filtering EM demonstrated that CCT-alpha bound to phosphorus-rich (phospholipid) structures in the glycogen. SP-B and SP-C, but not SP-A, localized predominantly to the glycogen stores. Collectively, these data suggest that the glycogen stores in fetal type II cells are a cellular site for surfactant PC synthesis and LB formation/maturation consistent with the idea that the glycogen is a unique substrate for surfactant lipids.     

The incorporation of isotopic carbon (14C) into the cerebral glycogen of rabbits

1. The incorporation of (14)C into the brain glycogen of conscious rabbits with labelled glucose, bicarbonate and glutamate as precursors has been studied. 2. Substantial incorporation from all these precursors was demonstrated after an interval of 5hr. from their injection. 3. With [(14)C]glucose maximal incorporation occurred at about 8hr. from the time of injection. 4. Hydrocortisone led to increased incorporation of (14)C from labelled glucose. 5. Some comparisons between the turnover of brain glycogen and that of skeletal and cardiac muscle are reported.