Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.     

Human platelet dense granules: improved isolation and preliminary characterization of [3H]-serotonin uptake and tetrabenazine-displaceable [3H]-ketanserin binding

An improved method for the isolation of human platelet dense granules was developed. A good yield (45%) of highly enriched (69-fold, based on serotonin content) dense granules was obtained after mild sonication and Percoll gradient centrifugation. The method has facilitated characterization of the granule, permitting the first report of Km and Vmax values for [3H]-serotonin uptake, as well as the first determination of Kd and Bmax values for tetrabenazine-displaceable [3H]-ketanserin binding, in the human platelet dense granule. The rates and affinities (Vmax 1.45 nmol/mg/min, Km 0.93 uM) of [3H]-serotonin uptake were similar to those previously reported for porcine dense granules. Tetrabenazine-displaceable [3H]-ketanserin binding was observed with a Kd (9.4 nM) similar to, and a Bmax (5.4 pmol/mg) approximately 10-fold lower than, that previously seen in bovine chromaffin granules.     

Leukophysin: a 28-kDa granule membrane protein of leukocytes.

A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.     

Dynamic histology of the antral epithelium in the mouse stomach: II. Ultrastructure and renewal of isthmal cells

The isthmus of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice using light and electron microscopy as well as 3H-thymidine radioautography. On the average, the isthmus measured 25 microns in length and was composed of 36 isthmal cells and two enteroendocrine cells. Isthmal cells generally displayed features found in embryonic cells, such as many free ribosomes, scant organelles, and a large reticulated nucleolus, and were, therefore, at an immature stage of development. Isthmal cells could be devoid of secretory granules ("granule-free cells," 2%) or contain a few small, spherical, PA-Schiff-positive, mucous granules in their apex. The granules in some of the cells had a variegated appearance and a diameter averaging 235 nm ("mottled granule cells," 39%); in other cells, the granules had a large diameter, 278 nm, with a pale background and a dense core ("core granule cells," 28%); while in still others they were homogeneously dark and measured 264 nm ("dense granule cells," 12%). Finally, some cells included a mixture of core and dense granules ("mixed granule cells," 14%). One hour after a single injection of 3H-thymidine, 37% of the isthmal cells were labeled. Each of the five isthmal cell types could acquire label and, therefore, divide. After one or more days of continuous 3H-thymidine infusion, all isthmal cells were labeled. Their turnover time was estimated to be 16.1 hr (t1/2 = 11.2 hr). The isthmus is thus composed of several cell types which are turning over rapidly. While all are relatively immature, the various types are thought to represent different developmental stages in the life history of an isthmal cell. A model devised on this basis proposes that the granule-free cells are stem cells, from which mottled granule cells are derived. These in turn evolve into either the dense granule cells of the upper isthmus or the core granule cells of the lower isthmus, or into the mixed granule cells (which are believed to develop eventually into dense granule cells or core granule cells). Maintenance of a steady state requires that the rapid production of isthmal cells be associated with rapid emigration; the dense granule cells presumably going to the pit and the core granule cells to the gland. The turnover of isthmal cells is accordingly described as following a "bidirectional pattern" of renewal.     

Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.     

An ultrastructural study of morphogenesis of fibrogranular complex and centriole in ductuli efferentes of Chinese hamster.

An ultrastructural study of ciliated epithelial cells in the ductuli efferentes of young and adult hamsters has revealed that these cells possess dense granules, dense granule clusters, dense bodies and fibrogranular complexes as reservoirs or precursors for ciliogenesis. The dense granules are first seen in the centrosomal region. Later, many dense granules and dense granule clusters appear in the apical portion of the epithelial cells where, subsequently, dense bodies are also found. Finally, the fibrogranular complexes are formed in adults. Morphological evidence strongly suggests that cilia are formed from diplosomal centrioles, de novo centrioles, dense body centrioles, and fibrogranular complex centrioles. Ciliogenesis begins in the fourth day after birth and increases rapidly in the fifth day. After the sixth day, cilia appear to be generated mostly from dense bodies and the total ciliogenesis activities gradually decrease as the animal ages.     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

Chromogranin B-induced secretory granule biogenesis: comparison with the similar role of chromogranin A

The two major proteins of secretory granules of secretory cells, chromogranins A (CGA) and B (CGB), have previously been proposed to play key roles in secretory granule biogenesis. Recently, CGA was reported to play an on/off switch role for secretory granule biogenesis. In the present study we found CGB being more effective than CGA in inducing secretory granule formation in non-neuroendocrine NIH3T3 and COS-7 cells. The mean number of dense core granules formed/cell of CGA-transfected NIH3T3 cells was 2.51, whereas that of CGB-transfected cells was 4.02, indicating the formation of 60% more granules in the CGB-transfected cells. Similarly, there were 55% more dense core granules formed in the CGB-transfected COS-7 cells than in the CGA-transfected cells. Moreover, transfection of CGA- and CGB-short interfering RNA (siRNA) into neuroendocrine PC12 cells not only decreased the amount of CGA and CGB expressed but also reduced the number of secretory granules by 41 and 78%, respectively, further suggesting the importance of CGB expression in secretory granule formation.     

The ultrastructure of the thyroid medullary carcinoma

The ultrastructure and the morphometrical pattern of secretory granules were studied in six cases of thyroid medullary carcinoma. The tumor cells were fusiform or polyhedral with irregular, mostly elongated nuclei. Phagolysosomes containing a crystalloid material, probably degraded lipoprotein complexes, degeneratively changed mitochondria, moderately developed rough endoplasmic reticulum and Golgi complexes were commonly found. Amyloid occurred as small fibrils in intercellular spaces. Marked dystrophic lesions of tumor cells surrounding amyloid fibrils were found. Numerous roundshaped electron-dense secretory granules were noticed in tumor cell cytoplasms. The morphometrical analysis showed that the size of granules oscillated between 60 and 450 nm with mean values ranging from 171.4 +/- 31.8 to 227.7 +/- 28.1 nm. Frequency distribution curves showed at least two peaks varying with the investigated case at different intervals. In two cases two distinct groups of granules were found within the same cells: one group of electron-dense, compact, smaller sized granules and another group of larger, finely granulated, less dense granules. In the other four cases the granule sizes were more homogeneous. These results might indicate that the granule size depends on the maturation degree and functional activity or that there are several kinds of granules specialized in the secretion of various substances.     

New class of cargo protein in Tetrahymena thermophila dense core secretory granules

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.     

Light and electron microscopic observations of fabrication, release, and fate of biphasic secretion granules produced by epididymal epithelial principal cells of the fan-throated lizard Sitana ponticeriana cuvier

The epididymis of the fan-throated lizard Sitana ponticeriana was examined with light and transmission electron microscopy to understand the cellular mechanisms of fabrication of secretion granules in epithelial principal cells, granule release into the lumen, and the fate of the dense structured granules after reaching the lumen. Principal cells of the ductus epididymis, except at the cauda, secrete electron-dense biphasic granules copiously, which decrease in abundance from the initial segment to corpus. The principal cell possesses a prominent Golgi apparatus and all versions of endoplasmic reticulum (ER), rough, smooth, and sparsely granulated. The material of the dense portion of the secretion granules, after processing at the Golgi apparatus, appears to accumulate in large ER cisternae in the supranuclear cytoplasm. It undergoes condensation when the cisternae become condensing vacuoles. Mitochondria appear to play a role in dense granule formation. The condensing vacuoles are displaced toward the apical cytoplasm when the material of the less dense portion is added to the condensing vacuoles at the Golgi area. Thus, the less dense and dense portions of the secretion granules are secreted and added to the condensing vacuoles separately. The composite granules are released into the lumen by exocytosis when the less dense portion merges with the luminal content, whereas the dense portion maintains its structured identity. The latter, initially measuring 1-2 microm in diameter, increases in size several times. It is inferred that these granules release their content gradually, resulting in the appearance of vacuoles, and suggesting that the granules have an insoluble matrix in which there is a sparingly soluble material. The substance leaching out of the granules appears to contribute to keeping the sperm quiescent and alive during storage in the male reproductive tract.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat

The expression and distribution of dense granule proteins in the enteric (coccidian) forms of Toxoplasma gondii in the small intestine of the cat. Experimental Parasitology 91, 203-211. The expression and location of the dense granule proteins (GRA1-6 and NTPase) in the merozoite and during asexual and sexual development of Toxoplasma gondii in the small intestine of the cat (definitive host) was examined by immuno-light and electron microscopy. This was compared with that of tachyzoites and bradyzoites present in the intermediate host. It was found that the merozoite contained the characteristic apical organelles plus a few large dense granules. By immunocytochemistry, dense granules in merozoites were negative for GRA proteins 1 to 6 in contrast to both tachyzoites and bradyzoites in which dense granules were positive for all six proteins. The GRA proteins were associated with the parasitophorous vacuole (PV) during tachyzoite and bradyzoite development but were absent from the PV of the enteric stages. However, the merozoite dense granules were positive for NTPase, which was similar to the tachyzoite while this antigen was down regulated in the bradyzoite. The apparent release of the NTPases into the PV formed by merozoites was also similar to that described for the tachyzoite, possibly reflecting the relative metabolic activity of the various stages. This study shows that the majority of GRA proteins have a similar stage-specific expression, which is independent of NTPases expression. These observations are consistent with T. gondii having a different host parasite relationship in the enteric forms, which does not involve the GRA proteins 1-6.     

Electron microscopy of human melanosomes in unstained,fresh air-dried hair bulbs and their examination by electron probe microanalysis

Summary Electron microscopy of unstained, fresh air-dried spreads of plucked human hair bulbs revealed relatively well preserved melanocytes and dense storage granules of melanin. Energy dispersive X-ray microanalysis of melanosomes and premelanosomes from black facial hair bulbs disclosed high peaks for potassium and sulfur, intermediate peaks for magnesium and calcium, and low but distinct ones for phosphorus and chlorine. No peaks for magnesium were found in the central portion of mature melanosomes, but at their edges and in premelanosomes conspicuous peaks for this element were obtained.Supported by a grant from the Ministry of Education of JapanThe author thanks Dr. H. Fujita for his advice and Messrs K. Yonehara, S. Okamura, T. Fukuoka, and T. Asai, Naka Works, Hitachi Ltd., Katsuta, Ibaraki, for use of their X-ray microanalyzer and for their assistance in electron probe analysis     

Diminished serotonin uptake in platelets of transgenic mice with increased Cu/Zn-superoxide dismutase activity.

Reduced levels of the neurotransmitter serotonin in blood platelets is a clinical symptom characteristic of individuals with Down's syndrome. To investigate the possible involvement of the Cu/Zn-superoxide dismutase (CuZnSOD) gene, which resides at the Down locus on chromosome no. 21, in the etiology of that symptom, we examined blood platelets of transgenic mice harboring the human CuZnSOD gene. It was found that platelets of transgenic CuZnSOD animals, which overexpress the transgene, contain lower levels of serotonin than nontransgenic littermate mice, due to a reduced rate of uptake of the neurotransmitter by the dense granules of the platelets. We found that the pH gradient (delta pH) across the dense granule membrane, which is the main driving force for serotonin transport, was diminished in dense granules of transgenic-CuZnSOD. Furthermore, a significantly lower than normal serotonin accumulation rate was also detected in dense granules isolated from blood platelets of Down's syndrome individuals. These findings suggest that CuZnSOD gene dosage is affecting the dense granule transport system and is thereby involved in the depressed level of blood serotonin found in patients born with Down's syndrome.     

Toxoplasma gondii ADP-ribosylation factor 1 mediates enhanced release of constitutively secreted dense granule proteins

Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424-2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPgammaS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter, Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.     

Proton NMR studies of nucleotide and amine storage in the dense granules of pig platelets

1H NMR measurements have been conducted at 360 MHz on isolated pig platelet dense granules. Resonances of the H8, H2 protons of the adenine ring, H1' protons of the ribose moiety, and the aromatic hydrogens of 5-hydroxytryptamine (5HT) have been identified in spectra of intact dense granules. Like the 31P resonances of the nucleotides contained in the dense granules (U?urbil et al., 1984), the line widths and the intensities of these resonances were sensitive to sample temperature and osmolarity of the suspension medium. Their chemical shifts indicate that 5HT in the granule interior is predominantly bound to the nucleotides through ring-stacking interactions. Association of 5HT with the nucleotides was also confirmed by the presence of intermolecular nuclear Overhauser effect (NOE) between 5HT and nucleotide protons. Large and negative intermolecular NOE's observed among the nucleotide H8, H2 and H1' protons, together with upfield shifts undergone by these protons within the dense granules, demonstrate that the nucleotides form a complex where they are in close proximity of each other. The formation of this complex apparently does not require the presence of amines since removal of 5HT and histamine did not change the chemical shifts of the nucleotide protons. From T1 and T2 data, rotational correlation time of 4 ns was calculated for the nucleotides in the dense granule interior at 35 degrees C. A resonance tentatively identified as H2 of histamine was found to shift upon manipulation of the intragranular pH; it was used as an indicator of pH changes within the granule interior during 5HT uptake and showed that 5HT accumulation increases the intragranular pH. These results demonstrate that 5HT is first taken up in response to the inside acidic pH gradient across the granule membrane and is subsequently sequestered in a matrix formed by the divalent cations and the nucleotides.     

Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.     

The effect of fixation conditions on the ultrastructural appearance of gastrin cell granules in the rat gastric pyloric antrum

The ultrastructural appearance of gastrin cell (G cell) granules was studied after different fixation procedures. When the pH of prefixation was varied there was greater preservation of the electron density of granule cores after acidic (pH 5.0 and 6.0) than after neutral or alkaline (pH 7.0 and 8.0) prefixation. Increasing duration of prefixation at pH 7.3 resulted in progressive loss of electron density of the granule core with swelling and occasional rupture of the limiting membrane. In tissues where most granules had been rendered electron lucent by fixation, those granules remaining dense cored were preferentially located close to the Golgi zone. These findings indicate that the electron density of G cell granules is profoundly affected by conditions of fixation, and that immature granules are more resistant to loss of core density than mature granules. They also suggest that the gastrin granule in vivo, like other polypeptide granules, may have a "solid", osmotically inactive core.     

EFFECTS OF IONOPHORE A23187 ON OOCYTES OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

Micromolar amounts of divalent cation ionophore A23187 stimulate full grown (but unfertilizable) oocytes of Comanthus japonica to undergo a cortical reaction that is incomplete: first, cortical granule contents ejected at exocytosis do not coalesce but remain as individual blebs just outside the oocyte; and, second, about a fourth of the cortical granule population does not undergo exo-cytosis and remains in the cortical cytoplasm. Of the cortical granules remaining in the oocyte, some have unreacted contents and others have morphologically modified contents. Fine structures are compared among unreacted cortical granules, internally-reacted cortical granules, extracellular blebs of cortical granule material and normal fertilization membranes. The comparison strongly suggests that the outer dense layer and inner fibrous layer of the normal fertilization membrane are derived, respectively, from the dense patches and from the matrices of the cortical granules.