Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.     

Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.     

Enzyme modulation of the Golgi apparatus and GERL: a cytochemical study of parotid acinar cells

The distribution of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) has been examined in resting parotid acinar cells as well as during decreased and increased secretory granule production. In resting acinar cells, TPPase activity was restricted to the trans Golgi saccules and AcPase activity was localized in GERL and immature secretory granules. Although secretory granule production is diminished during ethionine intoxication, no significant alteration in the distribution of either TPPase or AcPase was noted. However, marked changes in enzyme localization, especially of TPPase, occurred during accelerated secretory granule production. The alterations were essentially the same for all of the conditions studied (recovery from ethionine treatment, recovery from a protein depletion diet, secretory stimulation with isoproterenol, and postnatal maturation of the parotid gland). During maximal secretory granule production, TPPase activity was localized not only in the trans Golgi saccules, but also in GERL-like cisternae and immature secretory granules. The immature secretory granules were often in continuity with the GERL-like cisternae. At the same time that the TPPase activity was increased, the AcPase activity was frequently diminished. These modulations in enzyme activity provide evidence that GERL is derived from the trans Golgi saccule.     

Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.     

Membrane modification during secretory granule formation in rat somatotrophs

Somatotrophs from male rat anterior pituitary were used to investigate the formation of secretory granules. When enzymatically dispersed cells were incubated with cationized ferritin (CF) for 15 min, CF labeled immature secretory granules, but not mature granules of somatotrophs. Most immature granules labeled by CF transformed to the mature types within 120 min. This indicates that the fusion of endocytic vesicles with the immature granules occurs during the maturation process of secretory granules. The internalized CF was distributed not only in the immature secretory granules, but also in the peripheral region of trans Golgi cisternae or GERL. Enzyme cytochemistry revealed that acid phosphatase-positive cisternae (GERL) were the main site for secretory granule formation, and was devoid of thiamine pyrophosphatase (TPPase) activity. A small number of secretory granules were also present in the peripheral regions of TPPase-positive Golgi cisternae. The granule-forming sites, however, lacked TPPase activity, while the remaining region of the same cisterna showed the positive enzyme activity. This indicates that the granule-forming region at the periphery of Golgi cisterna is different from the remaining part of the same cisterna in terms of cytochemical properties. This probably results from the insertion of endocytic vesicle membrane, since the same granule-forming sites preferentially fused with CF-labeled small vesicles which lacked cytochemical TPPase activity. Taken together. Our results suggest that the membrane of secretory granules is modified during the granule formation, at least partly by the fusion of endocytic small vesicles with Golgi cisternae (or GERL), and with immature secretory granules.     

Fine structural localization of thiamine pyrophosphatase and acid phosphatase activities in the mouse pancreatic acinar cell

Summary The fine structural localization of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) was examined in pancreatic acinar cells of fasting and fed mice. The results were not affected by these conditions. TPPase activity was positive in two and sometimes three cisternae of the inner Golgi lamellae as well as in the condensing vacuoles of the trans area, but negative in the rigid lamellae and small vesicles of the trans area. AcPase activity was demonstrated in two and sometimes three cisternae of inner Golgi lamellae, condensing vacuoles, rigid lamellae, lysosomes and smooth or coated vesicles in the trans area. The inner Golgi lamellae and the condensing vacuoles were positive for both enzyme activities. From these facts, the lysosome is considered to be formed not only in the GERL system but also through the rough endoplasmic reticulum-Golgi apparatus route. It is reasonable to consider that Novikoff's GERL is not independent from the Golgi apparatus but represents a part of this organelle.This study was supported by a grant from the Japan Educational Ministry     

Morphological and cytochemical alterations of the Golgi apparatus and GERL in rat parotid acinar cells during ethionine intoxication and recovery

The present electron microscopic cytochemical investigation was undertaken to characterize the alterations in the golgi apparatus and GERL of rat parotid acinar cells during ethionine intoxication and recovery. Although the Golgi apparatus and GERL were reduced in size, and some broadening of the Golgi saccules occurred as the result of ethionine treatment, the relative localization of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules, and acid phosphatase activity (AcPase) in GERL, remained unchanged. Shortly after ethionine treatment was stopped, a dramatic redistribution of enzyme activities was noted. Within the first 24 hours of recovery, the Golgi apparatus began to enlarge, and the content of secretory granules increased. By day 3 of recovery, cisternae morphologically identifiable as GERL and forming secretory granules possessed TPPase activity, while AcPase activity was virtually undetectable. After seven days of recovery, the Golgi apparatus and GERL appeared both morphologically and cytochemically normal. The enzyme modulation observed during recovery may be correlated with increased secretory granule production. Furthermore, the presence of TPPase activity in GERL and forming secretory granules lends support to the suggestion that GERL may be derived from the trans Golgi saccule.     

Ultrastructural localization of cytoplasmic phosphatases in preimplantation mouse embryos.

The appearance and localization of the cytoplasmic phosphatases [acid phosphatase (AcPase) as a marker of lysosomes, TPPase as a marker of the Golgi apparatus, and NDPase (IDPase) as enzymatic marker of the endoplasmic reticulum (ER)] were cytochemically studied on the ultrastructural level in secondary oocytes and in preimplantation mouse embryos. The detectable AcPase activity, located on the inner surface of the membrane delimiting some cytoplasmic vacuoles (lysosomes and autophagic vacuoles), appears at the eight-cell stage and grows pregressively stronger up to the blastocyst stage. Golgi-associated reaction for TPPase was detectable in oocytes, dropped in one-cell embryos and became negative in the two-cell embryos. The reaction for TPPase and IDPase was present in plasma membranes of oocytes and early embryos and appeared in the delimiting membrane of some cytoplasmic vesicles in eight-cell embryos. Some activity of IDPase was found in small segments of the ER at the morula and blastocyst stage. The observed results suggest that the lysosomes are the first organelles in early embryos showing activity of the marker enzymes of the phosphatase type, while the activity of other marker enzymes is mainly concentrated in the plasma membrane of blastomeres. It cannot be excluded, however, that positive reaction for TPPase and IDPase in the plasma membrane results from nonspecific action of other phosphatases.     

Schistosoma mansoni: cytochemistry and morphology of the gastrodermal Golgi Apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.     

The relationships between the Golgi apparatus, GERL, and lysosomes of fetal rat liver Kupffer cells examined by ultrastructural phosphatase cytochemistry

Kupffer cells are the sinusoidal macrophages of the liver. Using ultrastructural phosphatase cytochemical methods, we examined the relationship between the Golgi apparatus, GERL, and lysosomes of Kupffer cells in fetal rat livers identified, in part, by their ability to phagocytize intravenously injected latex spheres. Thiamine pyrophosphatase (TPPase) activity was localized to the inner Golgi saccules and some vesicles in the Golgi region but not to GERL. A TPPase-like activity, demonstrable in lysosomes, was abolished by sodium fluoride but not suppressed by the alkaline phosphatase inhibitors L-cysteine and L-p-bromotetramisole. Acid phosphatase (AcPase) was localized by GERL, some coated vesicles, and in lysosomes, but not to the Golgi stacks. Continuities between GERL and lysosomes were observed. Phagosomes containing internalized latex spheres received TPPase and AcPase sequentially. TPPase was localized in phagosomes immediately after latex administration. AcPase activity was not found here until at least 10 minutes following the injection of the particulates. Our findings indicate that Kupffer cell lysosomes are derived from GERL, but also suggest that phagosomes may receive material packaged by the Golgi apparatus as well as GERL.     

Cytochemical studies on the internal polarity of the golgi apparatus and the relationship between this organelle and GERL

Summary Cytochemical studies were performed to clarify the occurrence of an internal polarity of the Golgi apparatus and the relationship between this organelle and GERL in many kinds of cells having different morphologies and functions. The fine structural localizations of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) were examined in anterior pituitary cells, thyroid epithelial cells, gastric chief and parietal cells, duodenal absorptive epithelial cells, hepatocytes, adrenal cortical and medullary cells of mice, and thyroid epithelial cells of domestic fowls. TPPase activity is usually localized in the cisternae of 1–3 stacks and vesicles on the trans-side of the Golgi apparatus of all the cells examined, and in some immature secretory granules of anterior pituitary cells and of gastric chief cells. Rigid lamellae and multivesicular bodies are rarely positive to this reaction, in several kinds of cells. AcPase activity was usually demonstrable in the cisternae of 1–3 stacks and vesicles on the trans-side of the Golgi apparatus, and also in rigid lamellae, coated vesicles, multivesicular bodies and lysosomes in all varieties of cells studied. Some immature secretory granules are positive to the AcPase reaction in anterior pituitary cells and gastric chief cells. The areas positive for both enzyme activities were partially or almost completely overlapping in all the cells examined, though there were minor variations among them. The grades of overlap are classified into three types. Prolonged osmication was performed on thyroid epithelial cells, duodenal absorptive epithelial cells, hepatocytes, adrenal cortical cells, Leydig cells, the epithelial cells of the vas deferens and the theca cells of mice. Cisternae of 1–3 stacks on the cis-side of the Golgi apparatus of all the cells examined were stained with osmium tetroxide. In all these cells we observed that the Golgi apparatus has an internal polarity and that GERL is a part of this organelle in cytochemical respects.This study was supported by grants from the Japan Ministry of Education     

Furrow formation in Xenopus embryos. Involvement of the golgi body as revealed by ultrastructural localization of thiamine pyrophosphatase activity.

The time-related ultrastructural distribution of thiamine pyrophosphatase (TPPase) activity was studied in first cleavage Xenopus embryos. In the furrow, localisation of the activity varied with the stage of development. The early furrow did not show any activity; however, when the furrow had progressed up to 300 μm into the embryo, activity was present at the furrow tip. With further development, heavy deposits were present in the furrow. In the cytoplasm, the activity was localised within the outermost cisternae of the Golgi body and in some of its associated vesicles. This distribution in the Golgi body prevailed irrespective of the stage of development. Vesicles and membrane cisternae elsewhere in the cytoplasm, especially near the furrow, were also positive. The demonstration of reaction product initially within the vesicles and cisternae of the Golgi body, and subsequently on the furrow surface, indicated that Golgi-derived, TPPase-positive vesicles might be adding membrane and surface material to the developing furrow.     

Cytodifferentiation in the Rana pipiens oocyte

Summary In early diplotene frog oocytes incubated to illustrate thiamine pyrophosphatase (TPPase) activity, reaction product is uniformly distributed within the compartments of the endoplasmic reticulum and nuclear envelope as well as within the saccules and small vesicles comprising the dictyosomes. With continued oocyte development the reaction product becomes concentrated in localized regions of the dictyosome saccules. Eventually, the enzyme is no longer apparent within the endoplasmic reticulum, but is concentrated in the cisternae of the inner dictyosome saccules. The variations noted suggest that the enzyme is synthesized early in diplotene by the endoplasmic reticulum and is subsequently transported to the Golgi apparatus where it is consistently observed at later developmental stages. TPPase activity is also present in the Golgi apparatus of follicle and theca cells as well as in ovarian epithelial cells. The enzyme is also detected in micropinocytotic vesicles contained within the cells comprising the follicle envelope and in intercellular spaces of the follicle. Horseradish peroxidase injected into the coelomic cavity is transported via micropinocytotic vesicles into and through the cells comprising the follicle envelope and in intercellular spaces. The exogenous protein is not found even after a prolonged time period in early diplotene oocytes. The protein is, however, present in large spherical and tubular vesicles in the cortex of vitellogenic oocytes approximately 500 microns in diameter. The possible functional role of the enzyme TPPase during oogenesis is discussed.This investigation was supported by a research grant from the National Science Foundation (GB-8736).     

The occurrence of the Golgi apparatus in mouse oocytes. Demonstration of thiamine pyrophosphatase activity

Location of thiamine pyrophosphatase activity as a marker of the Golgi apparatus was studied ultracytochemically in mouse oocytes with germinal vesicle (OGV), oocytes at metaphase I (OMI) and oocytes at metaphase II (OMII), and further in cells of the respective cumulus oophorus serving as comparative objects. TPPase activity in cumulus oophorus cells and in OGV was found exclusively in the Golgi apparatus. In OMI the reaction product of TPPase activity was observed in isolated smooth vesicles, and in only one case in structures identifiable as the Golgi apparatus. In OMII the occurrence of TPPase activity was also recorded in isolated smooth vesicles in cortical cytoplasm and further, exceptionally, in smooth concentrically arranged vesicles or tubules. The TPPase activity was not present in vesicular complexes. The results have shown that after the resumption of meiosis the occurrence of the reaction product of TPPase activity drops abruptly due to the reduction of the Golgi apparatus. Changes affecting the Golgi apparatus after the resumption of meiosis are related to the loss of the nucleus after the germinal vesicle breakdown.     

The localization of thiamine pyrophosphatase activity in Meckel's cartilage cells during endochondral ossification

The cytochemical distribution of thiamine pyrophosphatase (TPPase) activity in Meckel's cartilage cells of the mouse embryo has been studied during the endochondral ossification. All the cartilage cells contain reaction product within the Golgi apparatus. In immature chondrocytes, at the reserve cell zone, TPPase activity is restricted to several inner cisternae of independent Golgi apparatus. In mature cells at the proliferative cell zone, several Golgi complexes form a Golgi network connecting with each other by the TPPase positive tubular stalks. Golgi cisternae, condensing vacuoles and vesicles also contain reaction product. In the hypertrophic chondrocytes located in the calcifying zone, their disorganized Golgi apparatus still retain reaction product. Some chondrocytes, even those located within calcified or opened lacunae, exhibit intact structures and normal cytochemical enzyme distribution. These data indicate the possibility that some chondrocytes may survive and contribute the formation of mandible.     

The localization of thiamine pyrophosphatase activity in Meckel's cartilage cells during endochondral ossification

Summary The cytochemical distribution of thiamine pyrophosphatase (TPPase) activity in Meckel's cartilage cells of the mouse embryo has been studied during the endochondral ossification. All the cartilage cells contain reaction product within the Golgi apparatus. In immature chondrocytes, at the reserve cell zone, TPPase activity is restricted to several inner cisternae of independent Golgi apparatus. In mature cells at the proliferative cell zone, several Golgi complexes form a Golgi network connecting with each other by the TPPase positive tubular stalks. Golgi cisternae, condensing vacuoles and vesicles also contain reaction product. In the hypertrophic chondrocytes located in the calcifying zone, their disorganized Golgi apparatus still retain reaction product. Some chondrocytes, even those located within calcified or opened lacunae, exhibit intact structures and normal cytochemical enzyme distribution. These data indicate the possibility that some chondrocytes may survive and contribute the formation of mandible.     

Cellular compartments in mitotic cells: ultrahistochemical identification of Golgi elements in PtK-1 cells

This investigation focuses on the identification, distribution, and transport of intracellular membrane systems during mitosis. The membranes of the Golgi apparatus can be identified cytochemically by staining for acid phosphatase (acPase) and thiamine pyrophosphatase (TPPase) activity. Using this approach we are able to study the disintegration of the Golgi apparatus during mitosis and to follow the dislocation as well as the organized reappearance of Golgi elements after the completion of mitosis. We are able to demonstrate that during mitosis the activity of both enzymes is strong enough to react with the substrate applied during the staining procedure. Furthermore, we observe a characteristic pattern of membrane distribution in mitotic cells. During interphase the TPPase reaction is characteristically limited to one or two cisternae of a dictyosomal stack. The acPase reaction stains the membranes of the total stack, of the GERL, of some vesicles and cisternae near the dictyosomes and lysosomes. After the mitotic breakdown of the dictyosomal stacks the forming vesicles still stain positively and are distributed over the entire cytoplasm. At late anaphase and early telophase the enzyme activity occurs not only in the reconstituting dictyosomes but also in the nuclear envelope and in some ER cisternae. The extended spectrum of membrane structures indicating Golgi enzyme activity becomes obvious. This phenomenon favors the idea that at least some functions of the Golgi apparatus persist during mitosis.     

Colchicine-induced tubular,vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. I. Morphology and phosphatase cytochemistry

Treatment of rats with colchicine administered intraperitoneally at a dosage of 0.5 mg per 100 g of body weight for 6 hr induces extensive accumulations of tubular-vesicular and cisternal organelles in the absorptive cells of the small intestine. The formation of these organelle aggregates coincides with a reduction of microtubules and massive changes in the cellular organization including alterations of the Golgi apparatus and the plasma membrane. In most cases the accumulated tubules and vesicles contain a homogeneous electron-dense matrix, the cisternae often having the character of rigid lamellae. The organelle aggregates mainly occupy apical cell portions subjacent to the terminal web as well as basal cellular regions close to the basolateral plasma membrane. Tubular-vesicular as well as cisternal organelles react strongly for thiamine pyrophosphatase (TPPase), inosine diphosphatase (IDPase), acid phosphatase (AcPase) and trimetaphosphatase (TMPase). The staining pattern of TMPase differs from that of the other phosphatases in that the reaction is restricted to the colchicine-induced tubular-vesicular and cisternal aggregates, whereas TPPase, IDPase, and AcPase, respectively, also appear over Golgi stacks, multivesiculated bodies and plasma membrane. This phosphatase reactivity indicates the lysosomal character of the organelle aggregates.     

CYTOCHEMICAL STAINING OF MULTIVESICULAR BODY AND GOLGI VESICLES

To investigate the origin and nature of vesicles found within multivesicular bodies (mvb), the cytochemical staining properties of mvb vesicles were compared with those of other cytoplasmic vesicles, i.e. those associated with the Golgi complex and endocytic vesicles found near the apical cell surface. Rat epididymal tissue was stained in unbuffered OsO₄ for 40–48 hr, and the distribution of stain was compared to that of reaction products for acid phosphatase (AcPase) to mark lysosomal vesicles, or thiamine pyrophosphatase (TPPase) to mark certain Golgi vesicles, or infused with peroxidase (HRPase) to demonstrate endocytic vesicles. Mvb vesicles were stained only by OsO₄; AcPase, TPPase, and HRPase reaction products stained the mvb matrix. OsO₄ also stained certain vesicles along the convex surface of the Golgi complex. The findings suggest that mvb vesicles in epididymal epithelium are not lysosomes and are not involved in protein uptake. The majority of these vesicles have cytochemical reactions in common with vesicles located along the convex surface of the Golgi complex and may be derived therefrom. A minority are derived from the mvb-limiting membrane.