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.     

Fine structural localization of alkaline phosphatase in rat ovum during cleavage

On a submicroscopic level alkaline phosphatase activity was demonstrated by cytochemical methods in all stages of segmenting rat ova under survey, i.e. in the unfertilized and fertilized ovum, in the two-, four- and eight-cell stages and in the blastocyst. The reaction product was present in some cytoplasmic organelles as well as on cell membranes. A considerable number of cytoplasmic organelles with alkaline phosphatase activity was found in all stages from the one-cell up to the eight-cell stage. The reaction product was deposited in the tubules and vesicles of the smooth endoplasmic reticulum, in the nuclear envelope and in the Golgi complex as well. Some multivesicular bodies, autophagic vacuoles and majority of residual bodies out of the secondary lysosomes showed enzymatic activity. In the multicellular stages no significant differences were observed between the individual blastomeres in the incidence and distribution of the alkaline phosphatase activity. On the blastocyst-stage was found a low incidence of enzymatically active cytoplasmic organelles. Alkaline phosphatase activity was demonstrated in some minute vesicles below the cell membrane and in some secondary lysosomes. No essential differences were found between the cells of the embryoblast and the cells of the trophoblast in the incidence of enzymatically active structures. In the one-cell stage the activity of alkaline phosphatase was present on the cell membrane only sporadically, in the two- and four-cell stages enzymatic activity was found in this localization in a third of all specimen. In the eight-cell stage alkaline phosphatase activity was demonstrated on the cell membranes of all blastomeres. In the blastocyst the reaction product was deposited regularly on the membranes of the trophoblastic cells turned towards the zone pellucida, frequently on membranes of mutual tactile cells of the trophoblast and the embryoblast and only sporadically on cell membranes limiting the blastocyst cavity.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

Histochemical studies of the differentiation of microglial cells in the cerebral hemispheres of chick embryos and chicks

Summary Using histochemical procedures to reveal the presence, of nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase), we investigated the appcarance, distribution and ultrastructure of amoeboid and microglial cells in the cerebral hemispheres of chick embryos and young chicks, in order to clucidate the relationship between these two cell populations. On day 6 of incubation, a few round cells exhibiting NDPase, TPPase and AcPase activity were first detected in the thin mantle layer of the cerebral hemisphere. In the corpus striatum, these round cells increased rapidly in abundance until day 13 of incubation, after which their numbers gradually decreased, so that, on day 19 of incubation, they had entirely disappeared. Between day 10 and day 17 or 18 of incubation, round cells were located mainly in the zone of the mantle layer closest to the lumen. On day 10 of incubation, NDPase-, TPPase-and AcPase-positive cells that had a few short cytoplasmic processes (poorly ramified cells) were detected in the intermediate, and basal zones of mantle layer. They increased in abundance until day 17 or 18 of incubation and thereafter rapidly decreased in number. Round and poorly ramified cells exhibited NDPase activity on their plasma membranes and in their cytoplasmic vacuoles, with TPPase and AcPase activity being localized within their vacuoles. On day 19 of incubation, NDPase-and TPPase-positive cells with long, well-ramified cytoplasmic processes (well-ramified cells) were observed in the corpus striatum, these being mainly localized in the basal zone. After hatching, these cells increased rapidly in abundance and were distributed throughout the corpus striatum. These cells displayed NDPase and TPPase activity on their plasma membranes. These findings suggest that the round, the poorly ramified and the well-ramified cells belong to a single cell population.     

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.     

Histochemical studies of the differentiation of microglial cells in the cerebral hemispheres of chick embryos and chicks

Using histochemical procedures to reveal the presence of nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase), we investigated the appearance, distribution and ultrastructure of amoeboid and microglial cells in the cerebral hemispheres of chick embryos and young chicks, in order to elucidate the relationship between these two cell populations. On day 6 of incubation, a few round cells exhibiting NDPase, TPPase and AcPase activity were first detected in the thin mantle layer of the cerebral hemisphere. In the corpus striatum, these round cells increased rapidly in abundance until day 13 of incubation, after which their numbers gradually decreased, so that, on day 19 of incubation, they had entirely disappeared. Between day 10 and day 17 or 18 of incubation, round cells were located mainly in the zone of the mantle layer closest to the lumen. On day 10 of incubation, NDPase-, TPPase- and AcPase-positive cells that had a few short cytoplasmic processes (poorly ramified cells) were detected in the intermediate and basal zones of mantle layer. They increased in abundance until day 17 or 18 of incubation and thereafter rapidly decreased in number. Round and poorly ramified cells exhibited NDPase activity on their plasma membranes and in their cytoplasmic vacuoles, with TPPase and AcPase activity being localized within their vacuoles. On day 19 of incubation, NDPase- and TPPase-positive cells with long, well-ramified cytoplasmic processes (well-ramified cells) were observed in the corpus striatum, these being mainly localized in the basal zone. After hatching, these cells increased rapidly in abundance and were distributed throughout the corpus striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochemical study of the involvement of cell organelles in formation and accumulation of fibrillar amyloid in the pancreas of NORbeta transgenic mice

Phosphatase ultrastructural cytochemistry was used to evaluate the participation of cytoplasmic organelles in the accumulation of fibrillar amyloid beta (Abeta) in exocrine acinar cells and in macrophages of the pancreas of transgenic mice overexpressing a carboxy-terminal fragment of Abeta protein precursor (ABPP). Nucleoside diphosphatase (NDPase) and glucose-6-phosphatase (G6Pase) were used as cytochemical markers of the endoplasmic reticulum (ER), thiamine pyrophosphatase (TPPase) as a marker of the Golgi apparatus (GA), and acid phosphatase (AcPase) as a marker of lysosomes. Monoclonal antibody 4G8 raised against the 17-24 aa sequence of human Abeta protein was used for immunogold localization of fibrillar Abeta. The results of this study indicate that the formation of Abeta in acinar cells occurs directly in the vacuolar areas of the rough ER (RER) without evident participation of the elements of the GA, whereas an intimate structural relation with primary lysosomes suggests their role in modification or digestion of the deposited amyloid. In macrophages, fibrillar amyloid was present in numerous cytoplasmic vacuoles located frequently in close proximity to flattened saccules of the ER. This structural pattern revealed similarity to that observed previously in microglial cells producing fibrillar PrP amyloid in scrapie-infected mice and Abeta in brains of human elderly patients and in Alzheimer's type brain pathology.     

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.     

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.     

Ultrastructural localization of phosphatases in the testes of the domestic fowl: Acid phosphatase and thiamine pyrophosphatase

Summary ACPase and TPPase activity has been examined in the germinal epithelium of the testes in the domestic fowl. ACPase activity in spermatogonia and spermatocytes was confined to the Golgi complex. In spermatids ACPase activity was seen in the endoplasmic reticulum and nuclear envelope in the phase I and especially in the phase II (the elongating phase). This activity gradually decreased during the next phase III, and had disappeared in the final phase IV. The membrane body showed ACPase reaction in the small peripheral vacuoles and cisternal structures surrounding large central vacuoles. ACPase was also present in vesicles surrounding the developing tail. Late spermatids showed an abundance of autophagic vacuoles which had a complex array of ACPase positive delimiting membranes. In Sertoli cells ACPase activity was predominant in the lysosomes. TPPase activity was seen in the cisternae of the Golgi complex in spermatogonia and spermatocytes. In spermatids activity was present in the endoplasmic reticulum during the phase II, but it is lost in later stages. The smaller vacuoles and cisternal structures in the membrane body also showed reaction products. According to the present results it is thought likely that the smaller vacuoles and cisternal structures of the membrane body are of endoplasmic reticulum origin. The autophagic vacuoles in spermatids and the lysosomes of Sertoli cells are considered responsible for the degradation of residual bodies cast off by spermatids.     

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     

Structure and cytochemistry of the procambium in Salix buds during dormancy and dormancy breaking

This report presents a combined investigation of ultrastructural and enzymatic changes in the procambium from late winter to early spring. In January the procambial cells of dormant Salix buds have a convoluted plasma membrane with many plasmalemmasomes, numerous lipid bodies, large stacks of rough ER and plastids surrounded by smooth ER profiles. Several small lysosomes show activity of ATPase and acid phosphatases. In addition ER, nuclear envelopes, dictyosomes, and thylakoids have ATPase activity, and ER and plasmalemma, and nuclei also show acid phosphatase activity. In February metabolism seems to increase as indicated by lysosomes with membranous formations, dilated ER, nuclear envelopes, spiny vesicles, and polysomes. ATPase activity occurs in plasmalemma and vacuoles, and acid phosphatases in the middle lamella region of walls, in plasmalemma, vacuoles, ER, and nuclei. At the end of March, when growth starts inside the buds, but before they break, the stacks of rough ER disappear, and the vacuoles coalesce. Most of the lipid bodies have disappeared and the plastids have accumulated starch. Cell division and differentiation of procambial cells to protophloem and protoxylem have started. The distribution of ATPase increases; activity is found in walls and plasmalemma, and only a few small vacuoles still have ATPase and acid phosphatase activity. Notable is the appearance of ATPase in mitochondrial cristae and nucleoli and the occurrence of rather high levels also in endomembranes and dictyosomes.     

Density gradient separation of two populations of lysosomes from rat parotid acinar cells

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.     

Phosphatase activities and osmium reduction in cell organelles ofMicrasterias americana

Summary Organelles in resting and growing cells ofMicrasterias americana were examined using electron microscopy after cytochemical procedures for four kinds of phosphatases, acid phosphatase (ACPase), alkaline phosphatase (ALPase), thiamine pyrophosphatase (TPPase), and inosine diphosphatase (IDPase), and osmium tetroxide reduction. Special attention was paid to activities in the Golgi apparatus.In resting cells, positive reactions for ACPase and TPPase were observed in all cisternae of the dictyosome, especially in the peripheral parts. A positive IDPase reaction was seen in one central cisterna and was frequent in the distal-most cisterna. Reduction of osmium tetroxide was seen in the proximal cisternae.In early growing cells, the dictyosomes gave positive reactions for ACPase in the proximal cisternae and the distal cisterna, while in late growing cells only in proximal cisternae. Both in early and late growing cells, the dictyosomes were positive for TPPase and IDPase in the distal cisternae and vesicles derived from the distal cisternae, and for the reduction of osmium tetroxide in the proximal cisternae. ALPase activity was detected in the growing cell wall but not in the dictyosome.     

CYTOCHEMISTRY OF GOLGI FRACTIONS PREPARED FROM RAT LIVER

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF₁, GF₂, GF₃) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF₁ and GF₂, and along the outside of the cisternal membranes in GF₃. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF₁ and within many VLDL-filled vacuoles in GF₁ and GF₂, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF₃ and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF₁ and GF₂, and was not found in the cisternae in GF₃. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF₁ and GF₃, representing primarily trans-Golgi elements from the secretory Golgi face, and GF₃ consisting largely of cis-Golgi components from the opposite face.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

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.     

Ultrastructural cytochemistry of membrane-bound phosphatases in preimplantation mouse embryos.

The time of appearance and the ultrastructural localization of the enzyme activity of alkaline phosphatase (AlkPase), 5′-nucleotidase (5′Nuc), Mg²⁺-ATPase, transport ATPase, cyclic AMP phosphodiesterase (cAMP-PDase), and adenylate cyclase (AC) were investigated in unfertilized eggs and in mouse preimplantation embryos. Enzyme activity was associated only with the plasma membrane. AlkPase activity appeared only in limited areas of the plasma membrane of one-cell embryos and increased in the eight-cell and morula stages. In blastocysts, the enzyme activity was concentrated mainly in the trophoblast cells. 5′Nuc activity appeared first in four- or eight-cell embryos and the highest activity was observed in trophoblast cells in the blastocyst and in plasma membrane between cells forming inner cell mass. Mg²⁺-activated ATPase activity was present in all embryos and in unfertilized egg plasma membrane. Transport (Na⁺K⁺)-ATPase appeared only in the closely apposed membranes of adjacent cells in morulae and blastocysts. A very low cAMP-PDase activity appeared between adjacent cells in two-cell embryos, and the highest activity was observed on the outer surface of the plasma membrane of trophoblasts. AC was the only enzyme whose activity was located on the inner (cytoplasmic) side of the plasma membrane and appeared as early as the one-cell stage embryo. The relation between the time of the appearance of enzyme activity and the preparation of embryos for implantation and upon embryonic proliferative activity is discussed.     

Macrovacuolation induced by cytochalasin: its relation to the cytoskeleton; morphological and cytochemical observations

At higher doses of cytochalasin (e.g. 3 micrograms/ml for 3-20 hr), cells of the rat fibroblastoid line, Hmf, undergo extreme retraction, arborization, and subsequent rounding, and develop big cystic vacuoles. These vacuoles are always closely invested by microfilamentous masses, the CD-induced derivatives of the actin-based cytoskeleton, which aggregate in the endoplasm. Vacuolation is progressive (e.g. 12% cells at 6 hr; greater than 80% at 18 hr), related to total dose (concentration X time); and to congener (CD greater than CB). Vacuole membranes have the same dimension (85 A), surface marker 5'-nucleotidase, and junctional specializations as those found at the cell surface; they lack the membrane markers associated with endomembrane systems (e.g. AcPase, TPPase, IDPase) and are not lysosomal. Vacuoles represent internalized plasma membrane; they apparently result from retention in the endoplasm, and fusion, of pinocytotic vesicles originating at the cell surface. Vacuole membrane is always in intimate relation to the actin-based microfilament aggregates that surround the vacuoles, and actin-membrane linker proteins fodrin and vinculin are localized at the vacuole boundaries. Vacuoles and their enveloping actin-filament aggregates are surrounded by arrays of vimentin-based intermediate filaments. A new membranous compartment with characteristics of plasma membrane is thus formed within the cell under the influence of CD. Rounding brought about by other means causes no vacuolization. Macrovacuolation, like the other changes caused by CD, is completely reversible on restoration of cells to normal medium.