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

1968 ◽  
Vol 37 (2) ◽  
pp. 514-539 ◽  
Author(s):  
Everett Anderson
Keyword(s):  
Sea Urchin ◽  
Golgi Complex ◽  
Morphological Evidence ◽  
Cortical Granule ◽  
Perivitelline Space ◽  
Unit Membrane ◽  
Cortical Granules ◽  
Arbacia Punctulata ◽  
Cortical Reaction ◽  
Oocyte Differentiation

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.

scholarly journals 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

1970 ◽  
Vol 47 (3) ◽  
pp. 646-665 ◽  
Author(s):  
Frank J. Longo ◽  
Everett Anderson
Keyword(s):  
Sea Urchin ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Cell Stage ◽  
Zygote Nucleus ◽  
Arbacia Punctulata ◽  
Cortical Reaction ◽  
Female Pronucleus ◽  
Almost All ◽  
Granule Release

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.

scholarly journals Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

1996 ◽  
Vol 134 (2) ◽  
pp. 329-338 ◽  
Author(s):  
S S Vogel ◽  
P S Blank ◽  
J Zimmerberg
Keyword(s):  
Poisson Process ◽  
Sea Urchin ◽  
Physiological Responses ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
Calcium Dependence ◽  
Sea Urchin Egg ◽  
High Calcium ◽  
Cortical Granule Exocytosis

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

Cortical Granule Exocytosis and Fertilization Coat Elevation is Reduced in Aging Sea Urchin Eggs

2000 ◽  
Vol 6 (S2) ◽  
pp. 966-967
Author(s):  
Amitabha Chakrabarti ◽  
Heide Schatten
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Sea Water ◽  
Secretory Granules ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Lytechinus Pictus ◽  
Sea Urchin Eggs ◽  
Cortical Granule Exocytosis ◽  
Granule Secretion

Cortical granules are specialized Golgi-derived membrane-bound secretory granules that are located beneath the plasma membrane in unfertilized sea urchin eggs. Upon fertilization cortical granules discharge in a reaction induced by calcium and release their contents between the plasma membrane and a thin vitelline layer that lines the plasma membrane. Microvilli at the plasma membrane elongate incorporting cortical granule membranes during elongation. The vitelline layer elevates and becomes the egg's fertilization coat that hardens and serves as physical block to polyspermy. While we do not understand the precise mechanisms that participate in cortical granule discharge it is believed that actin plays a role in this process. Because actin and calcium metabolism is affected in aging cells we investigated if cortical granule secretion is affected in aging sea urchin eggs.Lytechinus pictus eggs were obtained by intracoelomic injection of 0.5M KCI to release the eggs into sea water at 23°C.

Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy

1995 ◽  
Vol 108 (6) ◽  
pp. 2293-2300 ◽  
Author(s):  
M. Terasaki
Keyword(s):  
Confocal Microscopy ◽  
Sea Urchin ◽  
Sea Water ◽  
Fluorescent Labeling ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Granule Exocytosis ◽  
New Methods ◽  
Cortical Granule Exocytosis ◽  
Fluorescent Dextran

A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1–43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs. The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

The relation between ionized calcium and cortical granule exocytosis in eggs of the sea urchin Echinus esculentus

1980 ◽  
Vol 207 (1167) ◽  
pp. 149-161 ◽  
Keyword(s):  
Molecular Mass ◽  
Electric Fields ◽  
Sea Urchin ◽  
Cortical Granule ◽  
Free Calcium ◽  
Relative Molecular Mass ◽  
Cortical Granules ◽  
Full Complement ◽  
Free Calcium Concentration ◽  
Cortical Granule Exocytosis

By subjecting sea urchin eggs to intense, short-duration, electric fields the permeability to low relative molecular mass substances is markedly increased. After such treatment, the extracellular space markers 22 Na + and [ 14 C]mannitol penetrate into the interior of the egg and localized destruction of the oolemma is apparent. The technique permits the rapid introduction of low relative molecular mass substances into the interior of the egg. We have employed it to investigate the efficacy of various buffered calcium concentrations in bringing about exocytosis of cortical granules of the egg. Eggs rendered permeable in the presence of EGTA (free Ca < 10 -8 M) retain a full complement of cortical granules and appear little different in cortical ultrastructure from unfertilized eggs, as judged by scanning and transmission electron microscopy. The proportion of cortical granules remaining in the egg cortex 30 s after application of an electric field in the presence of higher concentrations of calcium decreases pro­gressively as the free calcium concentration introduced into the egg interior is increased from 0.5 to 6 μM. The disappearance of the cortical granules is attributed to their having undergone exocytosis, since the changes in cortical ultrastructure that result from treatment with micro­-molar calcium concentrations are demonstrated to be similar to the changes that result from exocytosis of the cortical granules in intact eggs after fertilization.

233 STUDY OF CORTICAL GRANULE CONTENT IN IN VITRO-MATURED PORCINE OOCYTES BY MEANS OF PNA LECTIN PRECIPITATION

2008 ◽  
Vol 20 (1) ◽  
pp. 196
Author(s):  
M. D. Saavedra ◽  
M. Avils ◽  
P. Coy ◽  
R. Romar
Keyword(s):  
Polyvinylidene Fluoride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cortical Granule ◽  
Cortical Granules ◽  
Cortical Reaction ◽  
Agarose Beads ◽  
Sds Page ◽  
Pna Lectin

Cortical granules (CG) are clue organelles in the oocyte since their content is released under oocyte activation (i.e. fertilization) modifying the zona pellucida and thus blocking polyspermy. Once released, CG are not renewed. Research on cortical reaction and putative CG enzymes has progressed slowly because mammalian eggs contain only picogram quantities of CG-derived proteins (Moller and Wassarman 1989 Dev. Biol. 132, 103–112; Green 1997 Rev. Reprod. 2, 147–156), so the protein(s) responsible for the physiological changes in ZP after cortical reaction are not well known. The objective of this project was to study porcine CG content in in vitro-matured porcine oocytes by means of lectin precipitation with peanut agglutinin (PNA), since this lectin binds to porcine CG (Yoshida et al. 1993 Mol. Reprod. Dev. 36, 462–468). Immature porcine cumulus–oocytes complexes (COCs) from Landrace � Large White gilts were in vitro-matured for 44 h in NCSU-37 medium. After IVM period, COCs were stripped of cumulus cells, washed in PBS, and quickly washed through purified water. Then oocytes were lysed in a fresh water droplet by gentle pipetting using a narrow-bore glass pipette. Once lysed, zonae pellucidae were removed and oocyte cytoplasmic content (lysate) collected. Lysate from 1000 IVM-oocytes was incubated under continuous shaking (2 h, room temperature) with 100 µL PNA-agarose (Sigma, St. Louis, MO, USA) so that proteins bound to PNA could be precipitated by centrifugation. After lectin precipitation, proteins were detached from PNA-agarose beads by boiling in reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5 min, 100�C) (Laemmli 1970 Nature 227, 689). Samples were then centrifuged (5 min, 7000g), the pellet containing PNA-agarose beads was discarded, and the supernatant containing the proteins was collected and further separated by SDS-PAGE. The silver staining of electrophoresis gels revealed eleven bands from 37 to 180 kDa, so a second gel was electrotransferred to a polyvinylidene fluoride (PVDF) membrane (100V, 1 h) and incubated with PNA-horseradish peroxidase (PNA-HRP, 10 µg mL–1) for 1 h. Visualization was accomplished using the enhanced chemiluminiscence (ECL plus) method and Typhoon 9410 following the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany); only four bands of approximately 57 kDa, 60 kDa, 70 kDa, and 80 kDa were observed. These bands will be cut and processed for proteomic analysis for further studies. Preliminary results show that porcine CG-derived proteins can be studied by PNA lectin precipitation. These results could be employed in the future to develop specific antibodies against porcine CG.

Initiation of the cortical reaction in hamster and sheep oocytes in response to inositol trisphosphate

1988 ◽  
Vol 91 (1) ◽  
pp. 139-144
Author(s):  
D.G. Cran ◽  
R.M. Moor ◽  
R.F. Irvine
Keyword(s):  
External Medium ◽  
Cortical Granule ◽  
Dependent Manner ◽  
Cortical Granules ◽  
Cortical Reaction ◽  
Ph Dependent ◽  
Cortical Granule Exocytosis ◽  
Gamma S ◽  
Dose Dependent ◽  
External Ph

Microinjection of inositol 1,4,5-triphosphate into sheep and hamster oocytes induces secretion of cortical granules in a dose-dependent manner. In the sheep, this effect is strongly pH-dependent with minimal exocytosis taking place at pH 6.8 but a full cortical reaction occurring at pH8.0. Exocytosis in the hamster is also affected by the pH of the external medium but to a lesser extent. Injection of GTP gamma S also induces exocytosis in both species but is more effective in the hamster. It is suggested that inositol metabolism stimulated by sperm-egg interaction with a GTP-binding protein may be part of the mechanism leading to cortical granule exocytosis and that this may be modulated by the external pH.

Fertilisation in fish: a cortical alveolar lectin and its potential role in the block to polyspermy

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S66-S66 ◽  
Author(s):  
Shigeki Yasumasu ◽  
Nathan J. Wardrip ◽  
Bruce D. Zenner ◽  
Young M. Lee ◽  
Alan J. Smith ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Column Chromatography ◽  
Cortical Granule ◽  
Affinity Column ◽  
Cortical Granules ◽  
Amino Terminal ◽  
Sea Urchin Egg ◽  
Affinity Column Chromatography ◽  
Sperm Penetration ◽  
Carboxyl Terminal

An animal egg such as amphibian, mammalian or sea urchin egg receives only a single sperm at fertilisation. After binding of the first sperm, the egg is prevented from allowing the entry of additional sperm. In fact, polyspermy results in aborted development of the zygote. It has been generally accepted that a molecule(s) released from cortical granules participates in the block to polyspermy. As one such molecule, a cortical granule lectin has been isolated from unfertilised Xenopus eggs (Xenopus cortical granule lectin; XCGL). XCGL is released into the perivitelline space after fertilisation, and forms a complex with J1 jelly molecules to form an F layer, resulting in a block to additional sperm penetration.A lectin molecule has also been purified from the eggs of several species of fish. The fish egg lectin is located in the cortical alveoli and is released from them after fertilisation. However, its biological function is unclear. We isolated cortical alveolar lectin from unfertilised eggs of Chinook salmon through affinity column chromatography (salmon egg lectin; SEL). The lectin activity was estimated by haemagglutination. The activity of the purified SEL was most strongly inhibited by L-rhamnose and D-galactose, but not by EDTA. Further analysis by C4 reverse-phase column chromatography using HPLC revealed that the lectin was composed of three subunit proteins: 24K, 26Ka and 26Kb proteins. In addition, we cloned cDNAs for them by RT-PCR. The deduced amino acid sequence of the 26Ka protein was homologous with that of the 26Kb protein (identity, 96.4%). Identities of the 24K with the 26Ka and the 26Kb proteins were 55.9% and 66.7%, respectively. A database search revealed that a lectin molecule similar to the SEL had been identified in Anthocidaris crassispina egg (sea urchin egg lectin; SUEL). The SUEL is composed of 105 amino acids, and is similar to both amino-terminal and carboxyl-terminal halves of the SELs. Thus, the SEL molecule is composed of two repeats of such SUEL-like domains, suggesting that the SEL gene was produced by gene duplication.

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