The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae

Mannose-6-phosphate (Man-6-P) receptors for lysosomal enzymes were localized by immunocytochemistry in several secretory and adsorptive cell types using monospecific antireceptor antibodies. By immunofluorescence, the receptors were found in the Golgi region of polarized cells. When localized by immunoperoxidase at the electron microscope level, they were detected in Golgi cisternae, coated vesicles, endosomes, and lysosomes of all cell types examined (hepatocytes, exocrine pancreatic and epididymal epithelia). Within the Golgi complex, immunoreactive receptors were restricted in their distribution to one or two cisternae on the cis side of the Golgi stacks. They were not detected in trans Golgi or GERL cisternae. Based on their high concentration of Man-6-P receptors, we propose that the cis Golgi cisternae represent the site where the secretory and lysosomal pathways diverge: lysosomal enzymes bearing the Man-6-P recognition marker bind to Man-6-P receptors in this location and are delivered to endosomes and lysosomes via coated vesicles.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : II. Cytochemistry and Electron Microscopy of Bone Marrow Cells

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.     

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.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : I. Histochemical Staining of Bone Marrow Smears

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, β-galactosidase, β-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.     

INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL : I. Role of the Peripheral Elements of the Golgi Complex

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-¹⁴C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.     

Studies of the secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles

Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively. The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyelocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative. Our observations highly suggest that small Golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.     

Lysosomes in the cessation of vitellogenin secretion by the mosquito fat body; selective degradation of Golgi complexes and secretory granules

A massive and selective degradation of Golgi complexes, secretory granules, and RER is the mechanism responsible for the rapid termination of Vg secretion by trophocytes of the mosquito fat body. These cells are involved in an intensive synthesis of a glycoprotein, vitellogenin (Vg), which is accumulated by developing oocytes as yolk protein. Previously, assays for lysosomal enzymes have demonstrated that the cessation of Vg synthesis is characterized by a sharp increase in lysosomal activity; and fluorescent microscopy has shown that, during this intense lysosomal activity, Vg concentrates in lysosomes. In this report, electron microscopy combined with cytochemistry for lysosomal enzymes and localization of Vg with colloidal gold immunocytochemistry has shown that this lysosomal activity is directed towards selective degradation of Vg and organelles associated with its synthesis and secretion. Three organelles undergo lysosomal breakdown: the Golgi complex, Vg-containing secretory granules, and RER. The degradation of Golgi complexes occurs in two steps similar to that for RER: first, the organelle is sequestered by double isolation membranes, and the resulting pre-lysosome then fuses with a primary or secondary lysosome. In contrast, mature Vg-containing secretory granules fuse with lysosomes directly. This combination of crino- and autophagy is a specific, highly intense, and precisely timed event.     

Electron microscopic and cytochemical studies of the mouse subcommissural organ

Summary In mice most of the ependymal cells of the subcommissural organ (SCO cells) are densely packed with dilated cisternae of the endoplasmic reticulum (ER) containing either finely granular or flocculent materials. The well developed supra-nuclear Golgi apparatus consists of stacks of flattened saccules and small vesicles; the two or three outer Golgi saccules are moderately dilated and exhibit numerous fenestrations; occasional profiles suggesting the budding of coated vesicles and formation of membrane-bound dense bodies from the ends of the innermost Golgi saccules are seen. A few coated vesicles and membrane-bound dense bodies of various sizes and shapes are also found in the Golgi region.The contents of the dilated ER cisternae are stained with periodic acid-silver methenamine techniques. In the Golgi complex the two or three inner saccules are stained as deeply as the dense bodies, and the outer saccules are only slightly stained. The stained contents of ER cisternae are more electron opaque than those of the outer but less opaque than those of the inner Golgi saccules and the dense bodies.Acid phosphatase activities are localized in the dense bodies, some of the coated vesicles in the Golgi region, and in the one or two inner Golgi saccules.On the basis of these results the following conclusions have been reached: (1) In mouse SCO cells the finely granular and the flocculent materials in the lumen of ER cisternae contain a complex carbohydrate(s) which is secreted into the ventricle to form Reissner's fiber; (2) the secretory substance is assumed to be synthesized by the ER and stored in its cisternae, and the Golgi apparatus might play only a minor role, if any, in the elaboration of the secretory material; (3) most of the dense bodies in the mouse SCO cells are lysosomal in nature instead of being so-called dark secretory granules.Sponsored by the National Science Council, Republic of China.     

A non-autophagic pathway for diversion of ER secretory proteins to lysosomes

Intracisternal granules (ICG) develop in the rough ER of hyperstimulated thyrotrophs or thyroid hormone-secreting cells of the anterior pituitary gland. To determine the fate of these granules, we carried out morphological and immunocytochemical studies on pituitaries of thyroxine-treated, thyroidectomized rats. Under these conditions the ER of thyrotrophs is dramatically dilated and contains abundant ICG; the latter contain beta subunits of thyrotrophic hormone (TSH-beta). Based on purely morphologic criteria, intermediates were identified that appeared to represent stages in the transformation of a part rough/part smooth ER cisterna into a lysosome. Using immunocytochemical and cytochemical markers, two major types of intermediates were distinguished: type 1 lacked ribosomes but were labeled with antibodies against both ER markers (PDI, KDEL, ER membrane proteins) and a lysosomal membrane marker, lgp120. They also were reactive for the lysosomal enzyme, acid phosphatase, by enzyme cytochemistry. Type 2 intermediates were weakly reactive for ER markers and contained both lgp120 and lysosomal enzymes (cathepsin D, acid phosphatase). Taken together these results suggest that in hyperstimulated thyrotrophs part rough/part smooth ER elements containing ICG lose their ribosomes, their membrane is modified, and they sequentially acquire a lysosome-type membrane and lysosomal enzymes. The findings are compatible with the conclusion that a pathway exists by which under certain conditions, secretory proteins present in the ER as well as ER membrane and content proteins can be degraded by direct conversion of ER cisternae into lysosomes.     

Possible pathways for lysosomal enzyme delivery

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.     

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.     

Subcellular localization of complex carbohydrates in rat macrophages and monocytes.

Methods for visualization of complex carbohydrates ultrastructurally were employed to study specific organelles of the rat monocyte and macrophage. Vicinal glycols of glycoconjugates were demonstrated with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) postembedding sequence and acid groups were delineated by the dialyzed iron (DI) and high iron diamine (HID) preembedding techniques. Lysosomal bodies were generally found reactive with all three methods, although those of monocytes from the bone marrow and peripheral blood were notably lacking in acidic groups. The Golgi complex was consistently PA-TCH-SP-reactive, as were associated vesicles and occasional cisternal expansions, possibly related to GERL. Numerous cytoplasmic vesicles and small granulated structures and cisternae of the rough endoplasmic reticulum were also PA-TCH-SP-reactive.     

Multiple pathways of exocytosis, endocytosis, and membrane recycling: validation of a Golgi route

A number of pathways for intracellular membrane traffic have been detected in various cell types. The major established routes are: 1) the lysosomal pathway, which is the major route utilized in phagocytic and cultured cells; 2) the transcellular route, which represents the major type of traffic in nonfenestrated, capillary endothelial cells and which also appears to be the preferred route for the transport of immunoglobulins (intact) across cells; 3) the exocytosis pathway, utilized in secretory cells for discharge of secretory products, and which is also believed to be used for delivery of intrinsic membrane glycoproteins; 4) the plasmalemma to Golgi route, also highly developed in secretory cells, which is believed to be utilized for the recycling of secretory granule membranes; and 5) the biosynthetic pathways for transport of secretory products, lysosomal enzymes, and membrane proteins from the endoplasmic reticulum to the Golgi complex and for transport of lysosomal enzymes from the Golgi complex to lysosomes. It has become clear that cells repeatedly reutilize or recycle the membranes used in these various transport operations. Clathrin-coated vesicles have been found to be involved in transport along all these routes, which suggests that there are multiple populations of coated vesicles with different transport functions in every cell. It has become clear that the Golgi complex is the site where the membrane and product traffic converges and is sorted and directed to its correct destinations. The validation of a transport route from the cell surface to the Golgi complex raises the possibility that bound ligands and membrane constituents could be modified or repaired in transit during recycling through the Golgi complex, which is a biosynthetic compartment.     

Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach

Insulin and C-peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light-microscope level have detected both antigens in the same B cells. With the protein A-gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans-most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multigranular) structures. The fixation procedure used yielded excellent ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum-Golgi-granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans-cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis-side, those on the trans-side, and the trans-most rigid cisternae. Since insulin and C-peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C-peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C-peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C-peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.     

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

To investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest that endothelin is secreted by a constitutive pathway and that the lysosome may play an important role in regulating the biological activity of endothelin.     

PROTEIN SYNTHESIS,STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL : An Autoradiographic Study

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H³ as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H³; (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H³ can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at ∼5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at ∼20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.     

Quantitative aspects of membrane shifts in rat parathyroid cells initiated by decrease in serum calcium

The secretory activity of parathyroid glands in rats was stimulated by decreasing the serum Ca++ concentration through constant intravenous infusion of EGTA. The morphometric analysis of the nuclear and cytoplasmic volume and of the surface area of the rough endoplasmic reticulum, Golgi complex, secretory granules and plasma membrane revealed a membrane shift from secretory granules and Golgi complex to the plasma membrane within 1 hr of calcium depression. Subsequently, between 1 and 3 hr of calcium depression, the membrane shift was from the plasma membrane to the Golgi complex. It is considered likely that these membrane shifts are related to a rise in release of parathyroid hormone by exocytosis and a subsequent increase in retrieval of plasma membrane by endocytosis—probably through the compartment of coated pits and coated and uncoated vesicles.     

Further regression of seminal vesicles of castrated guinea pig by administration of cyproterone acetate

It has been established that a low level of secretory activity persisted in seminal vesicles of guinea pigs long after castration and that this may be due to a higher extratesticular androgen level in this animal. A RIA study revealed that the normal serum testosterone concentration of the guinea pigs was comparable to that of the rats, but the basal serum testosterone level after castration was ten times higher than rats under a similar condition. It was also shown that cyproterone acetate did not significantly lower the basal serum testosterone concentration in the castrated guinea pigs. The higher basal serum testosterone level is believed to be responsible for the slow and incomplete regression of this gland in the guinea pigs. There was a significant reduction in wet weight of the seminal vesicles after the treatment of castrated guinea pigs with cyproterone acetate. Ultrastructural study showed that there were both qualitative and quantitative changes in the cytoplasmic organelles. The Golgi apparatus further reduced in size and in the number of associated vesicles and vacuoles. There was a marked decrease in the number and size of secretory granules and lysosomes and an increase in the degree of undulation of the basement membrane. Accumulation of lipid droplets and glycogen was commonly observed. All these morphological evidences showed that further regression of the castrated guinea pig seminal vesicles can be achieved by cyproterone acetate treatment.     

The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules

Summary The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.