A FINE STRUCTURAL INVESTIGATION OF SURFACE SPECIALIZATIONS AND THE CORTICAL REACTION IN EGGS OF THE CNIDARIAN BUNODOSOMA CAVERNATA

Developing oocytes of the cnidarian Bunodosoma cavernata are located within the mesoglea of the mesenteries of the gastrovascular cavity. The cortex of the more mature vitellogenic oocytes contains numerous, electron-dense, membrane-bound, cortical granules. The surface of these oocytes possesses prominent radially projecting structures termed cytospines. Each cytospine has a core of microfilaments, 50–70 Å in diameter, that extends basally as a rootlet through the cortical layer. During spawning, ova lacking any extraneous investments are released from the enclosing gastrodermis. As a consequence of fertilization or events associated with the earliest stages of development the ova undergo a massive cortical reaction. This reaction, which occurs during or just after release of the ova, involves extensive reorganization of the cortical layer. The cortical granule membranes and egg surface membrane fuse and vesiculate resulting in the massive discharge of granule contents. This event is accompanied by the loss of vesicular remnants of cortical ooplasm and the disruption of cytospine organization. Light and electron microscope comparisons of unreacted and reacted eggs show that the reaction results in a significant decrease in egg diameter with the oolemma of the reacted egg reorganizing in a position centripetal to its original location.     

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.     

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.     

人精子甘露糖受体参与诱导卵母细胞皮质颗粒反应

目的研究人精子甘露糖受体与卵母细胞皮质颗粒反应的关系。方法以亲和层析法纯化的人精子甘露糖受体(purified mannose receptor,pMR)作用去透明带金黄地鼠卵,继而用罗丹明偶联的兵豆凝集素(Tetramethylrhodam ine Isothiocyanate Labeled Lentil,TRITC-LCA)标记。通过荧光显微镜观察被标记的皮质颗粒及卵母细胞表面甘露糖基的变化情况。结果pMR作用卵母细胞后,卵母细胞内的皮质颗粒减少且细胞表面的甘露糖基数量增加。结论pMR作用于卵母细胞表面后,可触发皮质颗粒反应,并使皮质颗粒部分内容物转移到卵母细胞表面。     

THE STRUCTURE AND FORMATION OF PROTEIN GRANULES IN THE FAT BODY OF AN INSECT

In the larva of the butterfly Calpodes ethlius, the fat body begins to store protein in the form of granules at about 30 to 35 hours before pupation, at a time when the endocuticle is being resorbed. At least two sorts of granule can be distinguished. The first granules to arise are those within vesicles of the Golgi complex. These may increase in size by incorporating material from microvesicles at their surface and by coalescence with one another. Later, at about 10 hours before pupation, another sort of granule arises by the isolation of regions of the endoplasmic reticulum (ER) within paired membranes derived from Golgi vesicles. Several of these ER isolation bodies coalesce, with fusion of their outer isolating membranes. The ribosomes and membranes may then disappear and the granules become indistinguishable from the protein granules formed from Golgi vesicles, or the ribosomes may remain and be embedded in dense crystalline protein, forming a storage body for both protein and RNA. Mitochondria are isolated within paired membranes in the same way as regions of the ER. The isolated mitochondria also coalesce in a similar manner. When the inner membranes are lost, the structure of a group of isolation bodies is indistinguishable from that of a cytolysome. Isolation within paired membranes, as described here, may be of general importance in segregating regions of massive lysis or massive sequestration.     

Ultrastructural observations of human and mouse oocytes treated with cryopreservatives

The effects of the cryopreservative agents dimethylsulfoxide (DMSO) and propanediol (PROH) on mature human and mature mouse oocytes have been examined with transmission electron microscopy. Treatment of CD-1 mouse oocytes and human preovulatory oocytes in a stepwise manner with either DMSO or PROH up to 1.5 M appears to trigger the exocytosis of 70-80% of the cortical granules in all oocytes. Successive stages in premature dehiscence, including a loss in granule electron density, fusion of the granule-limiting membrane with the oolemma, and extrusion of the cortical granule core into the perivitelline space, have been observed in all human oocytes studied. In addition, all human DMSO- and PROH-treated oocytes exhibited crypt-like invaginations and clusters of endocytic vesicles that subtend the oolemma. The presence of these crypts and pinocytotic vesicles in treated oocytes may suggest a mechanism for the retrieval of cortical granule membrane that is inserted into the original plasmalemma during exocytosis. The paucity of cortical granules in treated mouse and human oocytes as it potentially relates to an impaired ability to elicit the cortical reaction at fertilization is discussed.     

Preparation of the cortical reaction: maturation-dependent migration of SNARE proteins, clathrin, and complexin to the porcine oocyte's surface blocks membrane traffic until fertilization

The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles.     

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO₄. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (~800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (~500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.     

THE FINE STRUCTURE OF VON EBNER''S GLAND OF THE RAT

The fine structure of von Ebner's gland was studied in untreated rats and rats stimulated to secrete by fasting-refeeding or injection of pilocarpine. Cytological features were similar to those reported for pancreas and parotid gland. Abundant granular endoplasmic reticulum filled the basal portion of the cell, a well-developed Golgi complex was located in the vicinity of the nucleus, and the apical portion of the cell was filled with dense secretory granules. Dense heterogeneous bodies resembling lysosomes were closely associated with the Golgi complex. Coated vesicles were seen in the Golgi region and also in continuity with the cell membrane. Granule discharge occurred by fusion of the granule membrane with the cell membrane at the secretory surface. Successive fusion of adjacent granules to the previously fused granule formed a connected string of granules in the apical cytoplasm. Myoepithelial cells were present within the basement membrane, and nerve processes were seen adjacent to acinar and myoepithelial cells. Duct cells resembled the intercalated duct cells of the major salivary glands.     

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.     

SEGREGATION AND PACKAGING OF GRANULE ENZYMES IN EOSINOPHILIC LEUKOCYTES

During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins.     

STUDIES ON THE FINE STRUCTURE OF THE MAMMALIAN TESTIS : I. DIFFERENTIATION OF THE SPERMATIDS IN THE CAT (FELIS DOMESTICA)

The differentiation of cat spermatids was studied in thin sections examined with the electron microscope. The Golgi complex of the spermatid consists of a central aggregation of minute vacuoles, partially surrounded by a lamellar arrangement of flattened vesicles. In the formation of the acrosome, one or more moderately dense homogeneous granules arise within vacuoles of the Golgi complex. The coalescence of these vacuoles and their contained granules gives rise to a single acrosomal granule within a sizable membrane-limited vacuole, termed the acrosomal vesicle. This adheres to the nuclear membrane and later becomes closely applied to the anterior two-thirds of the elongating nucleus to form a closed bilaminar head cap. The substance of the acrosomal granule occupies the narrow cleft between the membranous layers of the cap. The caudal sheath is comprised of many straight filaments extending backward from a ring which encircles the nucleus at the posterior margin of the head cap. Attention is directed to the frequent occurrence of pairs of spermatids joined by a protoplasmic bridge and the origin and possible significance of this relationship are discussed.     

Characterization, fate, and function of hamster cortical granule components

Little is known about the composition and function of mammalian cortical granules. In this study, lectins were used as tools to: (1) estimate the number and molecular weight of glycoconjugates in hamster cortical granules and show what sugars are associated with each glycoconjugate; (2) identify cortical granule components that remain associated with the oolemma, cortical granule envelope, and/or zona pellucida of fertilized oocytes and preimplantation embryos; and (3) examine the role of cortical granule glycoconjugates in preimplantation embryogenesis. Microscopic examination of unfertilized oocytes revealed that the lectins PNA, DBA, WGA, RCA(120), Con A, and LCA bound to hamster cortical granules. Moreover, LCA and Con A labeled the zona pellucida, cortical granule envelope, and plasma membrane of fertilized and artificially activated oocytes and two and eight cell embryos. Lectin blots of unfertilized oocytes had at least 12 glycoconjugates that were recognized by one or more lectins. Nine of these glycoconjugates are found in the cortical granule envelope and/or are associated with the zona pellucida and plasma membrane following fertilization. In vivo functional studies showed that the binding of Con A to one or more mannosylated cortical granule components inhibited blastomere cleavage in two-cell embryos. Our data show that hamster cortical granules contain approximately 12 glycoconjugates of which nine remain associated extracellularly with the fertilized oocyte after the cortical reaction and that one or more play a role in regulating cleavage divisions.     

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm^?3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

CORTICAL CHANGES IN GROWING OOCYTES AND IN FERTILIZED OR PRICKED EGGS OF RANA PIPIENS

The folded cortex of the growing oocyte of the frog extends as microvilli into the substance of the developing vitelline membrane and, internal to the folds, possesses a layer of cortical granules. Free ribosomes, smooth-walled vesicles, coated vesicles, tubules, and electron-opaque granules are abundant in the peripheral zone of the cortex. Mitochondria, lipochondria, pigment granules, and electron-opaque granules are conspicuous between cortical granules and in the underlying endoplasm. Yolk platelets are restricted to the endoplasm. Cortical granules contain neutral and acid mucopolysaccharides, and possibly protein. In the mature oocyte, microvilli are withdrawn and the surface folds eliminated. Cortical granules now lie close to the plasma membrane, sometimes contacting it. Fertilization or pricking causes a wave of breakdown of cortical granules lasting 1–1½ min. Breakdown begins immediately after pricking but not until about 10–15 min after insemination, because the fertilizing sperm takes that long to penetrate the jelly and vitelline membrane. Cortical granules erupt through the surface and discharge their contents into the perivitelline space. Cortical craters left at sites of eruption soon disappear, and pseudopodial protrusions retract. By 30 min after insemination, the surface of the egg is relatively smooth.     

CELL MEMBRANE RESORPTION IN THE RAT EXOCRINE PANCREAS CELL AFTER IN VIVO STIMULATION OF THE SECRETION, AS STUDIED BY IN VITRO INCUBATION WITH EXTRACELLULAR SPACE MARKERS

Pancreatic secretion in the rat was stimulated in vivo by pilocarpine injection causing 90% of the storage granules to be discharged within 2 h. Incubation in vitro with [¹⁴C]sorbitol indicated that maximal ingestion of this extracellular space marker occurred 3 h after secretogogue injection. Morphological cell membrane measurements on cells with stimulated secretion revealed a simultaneous decrease in amount of membrane bordering the microvilli at the cell apex, lamellar processes, and infoldings present at the latero-basal face of these cells. In 3-h stimulated cells, having the average zymogen granule content characteristic for that phase of secretion, ferritin treatment in vitro showed that the infoldings and related fragmentation vesicles had ingested ferritin and could consequently be considered as being transport vehicles for redundant cell membrane. During stimulated secretion numerous vesicles and vacuoles appeared in the apical cytoplasm. Part of these structures were postulated to be related to the Golgi complex and were discussed in relation to secretory protein transport. Another part of these structures was assumed to have an endocytotic nature, although they never contained ferritin.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

Ultrastructural studies of differentiation in the oocyte of the polyclad turbellarian,Prostheceraeus floridanus

Oocyte differentiation in the polyclad turbellarian Prostheceraeus floridanus has been examined to determine the nature of oogenesis in a primitive spiralian. The process has been divided into five stages. (1) The early oocyte: This stage is characterized by a large germinal vesicle surrounded by dense granular material associated with the nuclear pores and with mitochondria. (2) The vesicle stage: The endoplasmic reticulum is organized into sheets which often contain dense particles. Vesicles are found in clusters in the cytoplasm, some of which are revealed to be lysosomes by treatment with the Gomori acid phosphatase medium. (3) Cortical granule formation: Cortical granules are formed by the fusion of filled Golgi vasuoles which have been released from the Golgi saccules. The association between the endoplasmic reticulum and Golgi suggests that protein is synthesized in the ER and transferred to the Golgi where polysaccharides are added to form nascent cortical granules. (4) Yolk synthesis: After a large number of cortical granules are synthesized, yolk bodies appear. They originate as small membrane-bound vesicles containing flocculent material which subsequently increase in size and become more compact. Connections between the forming yolk bodies and the endoplasmic reticulum indicate that yolk synthesis occurs in the ER. (5) Mature egg: In the final stage, the cortical granules move to the periphery and yolk platelets and glycogen fill the egg. At no time is there any evidence of uptake of macromolecules at the oocyte surface. Except for occasional desmosomes between early oocytes, no membrane specialization or cell associations are seen throughout oogenesis. Each oocyte develops as an independent entity, a conclusion supported by the lack of an organized ovary.     

GROWTH AND DIFFERENTIATION OF CYTOPLASMIC MEMBRANES IN THE COURSE OF LIPOPROTEIN GRANULE SYNTHESIS IN THE HEPATIC CELL : I. Elaboration of Elements of the Golgi Complex

The synthesis of "very low" density lipoprotein in liver cells is characterized by the fact that the synthesized products, mostly triglycerides, are processed in the form of discrete, size-limited granules or globules, about 400 A in diameter. The present investigation has been made possible in part by the use of a fixative (OsO₄ in bidistilled H₂O at pH 6.0, in the absence of electrolytes) particularly effective in preserving cytoplasmic membranes and lipids, and giving them high stainability and differential contrast. Under these technical conditions, the lipoprotein granules retain their morphology and high density to electrons practically unaltered, and may serve as tracers in determining their route of transport from the sites of synthesis, starting at the rough-smooth ER junctions, to the lumen of Golgi concentrating vesicles. From the observations, it may be deduced that, along with lipoprotein granule synthesis and transport, there are also production and transfer of new membranes in the form of tubular extensions of smooth ER network which, by progressive fusion and coalescence, participate in the elaboration of fenestrated plates and solid Golgi sacs. In contradistinction to the entire process of liver lipoprotein granule synthesis, transport, and segregation, as reported in the present paper, appears to constitute a developmental sequence which includes the following communicating compartments, in consecutive order: cisternae of rough ER where proteins and possibly phospholipids are synthesized, smooth ER network where triglycerides are synthesized and transported in the form of dense granules, fusion of smooth ER tubular extensions into Golgi fenestrated plates, and further coalescence into solid Golgi sacs, ending in the segregation of the granules in appended concentrating vesicles, or detached "secretory vesicles." It seems that it is this progressive evolution in growth and configuration of membranes which is reflected in the so called polarity, from forming to mature faces, of the Golgi apparatus.