Characterization of the proteolytic compartment in rat hepatocyte nodules

Persistent liver nodules (hepatocyte nodules, neoplastic nodules) were produced in rat liver by intermittent feeding with 2-acetylaminofluorene. Dense bodies (secondary lysosomes) were purified and characterized from the nodules. The purity of the dense body fraction was 90%. The levels of various lysosomal enzyme activities were lower in these dense bodies in comparison with dense bodies from control liver. Similarly, protein degradation was 50% lower in dense bodies from liver nodules than in control liver. The number of autophagic vacuoles (AVs) in the nodular tissue increased considerably after 3 h vinblastine treatment. We have taken advantage of this expansion in an effort to isolate these organelles from liver nodules. Autophagic vacuoles have been isolated recently from liver and kidney but not from putatively premalignant liver nodules. Fraction purity of AVs from liver nodules was 95%. As with dense bodies, AVs from nodular tissue displayed lower activities of proteinases and lower rates of protein degradation when compared with their counterparts from normal liver tissue. Accordingly, the lower rate of overall protein degradation in liver nodules can be ascribed to a decrease in lysosomal activity. A diminished autophagic sequestration capacity is the most plausible explanation for the decreased rate of proteolysis in cells. This could conceivably give these nodular cells a growth advantage and assist in their selective outgrowth as well as in their transformation from neoplastic into true cancer cells.     

QUANTITATIVE CHARACTERIZATION OF DENSE BODY, AUTOPHAGIC VACUOLE, AND ACID PHOSPHATASE-BEARING PARTICLE POPULATIONS DURING THE EARLY PHASES OF GLUCAGON-INDUCED AUTOPHAGY IN RAT LIVER

Quantitative characterization of dense body, autophagic vacuole and acid phosphatase-bearing particle populations of rat liver have been made at 10 min intervals during the first 50 min following the intraperitoneal administration of glucagon. Beginning 10 to 20 min postinjection, increases in the number of autophagic vacuoles and in the osmotic sensitivity of acid phosphatase-bearing particles were observed, associated with a progressive disappearance of dense bodies. These changes appeared to reach a maximum 50 min after treatment. The average volume of autophagic vacuoles was found to be 440–870% greater than that of normal dense bodies during this time period. No consistent change in total acid phosphatase activity was noted. A detailed study of autophagic vacuole profile populations revealed the presence of five different types of profiles, two of which, types I and II, accounted for 76.3–94.4% of the profiles examined. Type I profiles primarily contained elements of the endoplasmic reticulum, free ribosomes, and ground cytoplasm. Type II profiles had mitochondrial profiles as their principal constituent, but endoplasmic reticulum and free ribosomes were also seen. At all time points type I profiles predominated, comprising 55–69% of the profiles found. Both profile types were bounded by single and double limiting membranes, the former being predominate. A time-dependent change in the ratio of single to double membrane-limited profiles could not be demonstrated. Morphometric parameters derived from profile size distributions indicated that the number of types I and II autophagic vacuoles increased with time, the rate being greater for the type II particle, except between 40 and 50 min. The average volume of the type II autophagic vacuole was consistently greater than that of the type I.     

Distribution of Acid Phosphatase in Paramecium caudatum: Its Relations with the Process of Digestion

SYNOPSIS. The distribution of acid phosphatase was investigated at the ultrastructural level in Paramecium caudatum. Acid phosphatase occurs in endoplasmic reticulum, Golgi apparatus, food vacuoles, autophagic vesicles, vacuolar and dense bodies. Some slight deposits are also seen in the mitochondria.
These observations point out that this hydrolase activity is related to digestive processes. The enzyme, originating from the endoplasmic reticulum and Golgi apparatus reaches the food vacuole or autophagic vesicle likely via the reticulum. The digestion of the bacteria or of the enclosed organelle gives rise to electronopaque material which is later found in dense bodies. These dense bodies are likely secondary lysosomes and it is possible that they may fuse with the young food vacuole or with autophagic vesicles.     

Analog modeling of glucagon-induced autophagy in rat liver. II. Evaluation of iron labeling as a means for identifying telolysosome, autophagosome and autolysosome populations.

To evaluate the potential usefulness of iron labeling as a means for identifying the telolysome, autophagosome and autolysosome populations of rat liver, animals treated with Jectofer (iron-citric acid-sorbitol complex), or with Jectofer followed by glucagon, have been studied with a variety of biochemical and morphological methods. Differential centrifugation studies of liver homogenates revealed that the sedimentation velocity and mechanical fragility of acid phosphatase bearing particles increase with the duration of Jectofer treatment and that iron accumulates in the mitochondrial and nuclear fractions. Rate sedimentation studies confirmed the change in sedimentation velocity, which was shown to be due in part to a marked increase in particle density. Quantitative morphological analysis of liver M + L and N + M + L fractions revealed a nearly complete absence of pericanalicular dense bodies after 6–7 days of Jectofer treatment. In these fractions a new type of particle containing fine electron dense granules was seen. The mean volume of these particles was decreased and their number increased when compared to dense bodies but the general morphology and overall size distribution of the two particle classes were similar. In animals given both Jectofer and glucagon, autophagic vacuole formation was similar to that found in animals receiving only glucagon. However, the increase in osmotic fragility of acid phosphatase bearing particles usually seen after glucagon administration occurred at a significantly slower rate. Examination of paniculate fractions revealed the presence of autophagic vacuoles with (autolysosomes) and without (autophagosomes) fine dense granules. The number of autolysosomes and their relative proportion in the autophagic vacuole population were correlated with an increase in the osmotic fragility of the acid phosphatase bearing particles in the same fraction. Organelle degeneration was observed more frequently in autolysosome profiles. These results support the contention that iron labeling can be used to separate the principal particle populations participating in the autophagic response induced by glucagon.     

Ultrastructural Localization of Acid Phosphatase in Starved Tokophrya infusionum*

SYNOPSIS. Young organisms of Tokophrya infusionum starved for several hr, are best suited for a study of the fine structure of this organism including the distribution of its organelles. Acid phosphatase was localized by a combined electron microscopy and cytochemical approach using modified Gomori methods. The enzyme was found in small dense bodies, spheroid vesicles, missile-like bodies, rough-surfaced endoplasmic reticulum, residue and autophagic vacuoles. The small dense bodies are thought to be primary lysosomes since electron micrographs show a) a continuity between the membrane of the rough-surfaced endoplasmic reticulum and that of the dense bodies and b) a connection between the contents of both structures when the dense bodies form from the endoplasmic reticulum.     

STUDIES ON THE INTRACELLULAR DIGESTIVE PROCESS IN MAMMALIAN TISSUE CULTURE CELLS

DNA-protein coacervates containing colloidal gold particles were readily phagocytized by strain L fibroblasts. During the subsequent digestion process, the gold particles served as markers which permitted the demonstration of the evolution of digestive vacuoles to multivesicular bodies and finally to dense bodies. Acid phosphatase and esterolytic activity was present in these structures. The hydrolytic enzymes were apparently brought to the phagocytotic vacuoles in small vesicles originating in the Golgi region. These vesicles entered the vacuoles prior to the digestion of the coacervates and the appearance of positive cytochemical reactions. The cytoplasmic dense bodies frequently merged with the phagocytotic vacuoles. This was demonstrated by prelabeling the dense bodies with colloidal iron prior to phagocytosis of the coacervates. In addition, evidence is presented for the interrelationship of the phagocytotic and autophagic pathways.     

Autophagocytosis in the larval midgut cells of Pieris brassicae during metamorphosis

Summary We observed three types of cells in the epithelial layer of the midgut of last instars of Pieris brassicae. The columnar and goblet cells degenerate during the second part of the last larval stage while the undifferentiated basal cells proliferate during this period and create the epithelium of the pupal midgut. The first morphological sign of involution is the formation of autophagic vacuoles and dense bodies in the cytoplasm of columnar and goblet cells which begins on day 4 of the stage. The number and size of autophagic vacuoles and dense bodies increase during the spinning period (85–96 h). Finally, at the end of the stage, the columnar and goblet cells become displaced by the growing pupal epithelium and reach the lumen where they disintegrate.Autophagocytosis was not seen in the cells during the feeding period (0–72 h). However, we observed many autophagic vacuoles in the columnar and goblet cells of 50-h-old instars 3 h after the administration of 30 g/g body weight of 20-hydroxyecdysone. The hormone treatment elevated by 100% the incorporation of ³H-leucine into the proteins of the midgut. Inhibitors of protein synthesis, cycloheximide and puromycin, in doses that supressed the incorporation of the amino acid by 60–70% either in hormone treated or untreated larvae, exerted diverse effects on the autophagic process. Puromycin did not block the hormone-induced formation of autophagic vacuoles while cycloheximide prevented it. Possible explanations for this diversity are discussed.     

Significance of electron dense microbodies in trap cells of the nematophagous fungus Arthrobotrys oligospora

We have studied the fate of electron dense microbodies in nematode-trapping organs (traps) of the fungus A. oligospora during the initial hours following nematode capture. The interaction studies were performed with isolated traps which had captured a nematode under conditions where the fungal cells had no access to external energy sources. Video enhanced contrast microscopy showed that under these conditions the number of dense bodies present in the trap cell that formed the penetration tube, rapidly decreased. During subsequent penetration and development of the infection bulb this decrease continued while at this time common cell organelles such as mitochondria and vacuoles were formed. This was confirmed by electron microscopy which also revealed that the dense bodies were degraded by means of an autophagic process. The organelles were degraded individually and finally turned into compartments which, based on ultrastructural criteria, were considered vacuoles. Fusion of such vacuoles into larger organelles frequently occurred. The degradation process was initiated early in the interaction since initial stages were already evident within 15 min after capture. Generally it took 1–2 h before the infection bulb had fully developed and trophic hyphae formation started. During this time the original trap cell, characterized by numerous dense bodies, was transformed into an active vegetative hyphal cell containing typical cell organelles such as nuclei, mitochondria, a strongly proliferated endoplasmic reticulum, vacuoles and normal microbodies but lacked dense bodies. This disappearance of dense bodies was confined to the cell that penetrated the nematode and—less frequently—its two neighbouring cells in the hyphal loop. In the other cells, constituting the trap, the dense bodies remained unaffected. As will be discussed, the present results support our current view that traps of A. oligospora contribute to the survival of the organism in its natural environment.     

Morphology of the neural lobe of the hypophysis in rats treated with furosemide. I. Neurosecretory axons

The changes found in the neurosecretory axons of the neural lobe of the hypophysis were studied in rats treated with furosemide for three days: over the whole period of treatment, the animals, according to each group, were deprived or not of water. In the animals with free access to water the axons contained neurosecretory granules with scarce content and low electron density; only some of them had vacuoles and autophagic bodies. In the animals deprived of water axon morphology was variable and axons showing vacuoles and autophagic bodies were abundant, as well as those presenting lamellar and dense bodies and also those in which filaments prevailed. The most outstanding modifications in this last group of animals were related to the dehydration and were not found in those animals which were given access to water again and which were in consequence rehydrated.     

多泡体形成过程的细胞化学研究

Multivesicular bodies were observed frequently in electron microscope photographs of Leydig cells from normal adult rat testes. Their formation, evolution and fate were analyzed morphologically in preparations treated to show cytidine monophosphatase (CMPase) activity and in animals sacrificed at various time intervals ranging from 5 min to 2 hrs after a single intratesticular injection of cationic ferritin (CF). Analysis of morphological and cytochemical data led to the following interpretation for the origin and fate of the multivesicular bodies in Leydig cells. The formation of multivesicular bodies in Leydig cells can be divided into three steps. Step 1, some endocytic vacuoles in Golgi region fuse with small vesicles to form pre-multivesicular bodies. Step 2, the pre-multivesicular bodies fuse together to form pale multivesicular bodies which are characterized by their large size, pale matrix and paucity of internal vesicles. Step 3, the pale multivesicular bodies remove their surplus enveloping membrane to become dense multivesicular bodies which are characterized by their smaller size, dense matrix and filling with internal vesicles. The pre-multivesicular bodies and pale multivesicular bodies do not contain hydrolytic enzymes, the dense multivesicular bodies acquire their hydrolytic enzymes by fusion with lysosomes and show CMPase activity. The dense multivesicular bodies often show a very close association with autophagosomes, and they might be involved in the autophagic activity of Leydig cells.     

多泡体形成过程的细胞化学研究

本实验用酶细胞化学和示踪细胞化学方法观察了睾丸间质细胞中多泡体的形成过程及其与溶酶体的关系。实验结果表明,睾丸间质细胞中多泡体的形成可分三个阶段:首先,一些含内吞物质的泡状结构进入高尔基体区域,与那里的小泡融合,形成内含少量小泡的前多泡体;然后,前多泡体互相融合,形成体积较大、基质电子密度低、内含小泡排列稀疏的低电子密度多泡体;最后,低电子密度多泡体通过表面长出微绒毛样结构并不断断裂的方式去除多余的界膜,形成体积较小、基质电子密度高、内含小泡排列紧密的高电子密度多泡体。因此,多泡体的形成既与内吞活动有关,又与高尔基体区域小泡有关。前多泡体和低电子密度多泡体不含溶酶体酶。在多泡体形成过程中,只有到高电子密度多泡体阶段,才与溶酶体发生关系,从溶酶体中获取溶酶体酶。多泡体形成后,常与自体吞噬泡靠近,可能参与睾丸间质细胞的自体吞噬活动。     

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.     

Analysis of autophagy in Aspergillus oryzae by disruption of Aoatg13, Aoatg4, and Aoatg15 genes

Autophagy is a degradation system in which cellular components are digested via vacuoles/lysosomes, and involved in differentiation in addition to helping cells to survive starvation. The autophagic process is composed of several steps: induction of autophagy, formation of autophagosomes, transportation to vacuoles, and degradation of autophagic bodies. To further understand autophagy in the filamentous fungus Aspergillus oryzae, we first constructed A. oryzae mutants defective for the Aoatg13, Aoatg4, and Aoatg15 genes and examined the resulting phenotypes. The ΔAoatg13 mutant developed conidiophores and conidia, although the number of conidia was decreased compared with the wild-type strain, while conidiation in the ΔAoatg4 and ΔAoatg15 mutants was not detected. The ΔAoatg15 mutants displayed a marked reduction of development of aerial hyphae. Moreover, autophagy in these mutants was examined by observation of the behavior of enhanced green fluorescent protein (EGFP)-AoAtg8. In the ΔAoatg13 mutant, the slight accumulation of EGFP-AoAtg8 in vacuoles, preautophagosomal structures (PAS), and autophagosomes was observed, whereas only PAS-like structures were detected in the ΔAoatg4 mutant. In the ΔAoatg15 mutant, autophagic bodies accumulated in vacuoles, suggesting that the uptake process proceeded. We therefore propose that the level of autophagy is closely correlated with the degree of differentiation in A. oryzae.     

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse

1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediately after 3 hr feeding period. 2. The size of the autophagic vacuole and lysosome (dense body) compartments increased in both types of cells during 2-72 hr fasting parallel with decreasing serum insulin levels. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.     

LYSOSOME FUNCTION IN THE REGULATION OF THE SECRETORY PROCESS IN CELLS OF THE ANTERIOR PITUITARY GLAND

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH¹ was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.     

Hepatic autophagy and intracellular ATP. A morphometric study

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.     

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.     

A MORPHOMETRIC STUDY OF THE REMOVAL OF PHENOBARBITAL-INDUCED MEMBRANES FROM HEPATOCYTES AFTER CESSATION OF TREATMENT

It is well known that phenobarbital (PB) treatment produces an increase in the amount of cytoplasmic membranes of hepatocytes, with a parallel enhancement in the activity of drug-metabolizing enzymes. However, little is known about how the induced membranes are removed after the drug treatment is stopped. To consider this problem, the recovery of rat hepatocytes from PB induction (five daily injections, 100 mg/kg) was followed morphometrically. Treatment with PB produced a cellular enlargement (26%) due to increases in the volume of the cytoplasmic matrix (20%) and the volume (100%) and surface area (90%) of the smooth-surfaced endoplasmic reticulum (SER). The volume of the nuclei and the surface area of the Golgi apparatus were also increased, but no changes were detected in the volumes of the mitochondria or peroxisomes. The SER membranes induced by the PB were removed within 5 days after the end of the treatment period. During this period of membrane removal, we observed an increase in the volume (800%) and number (96%) of autophagic vacuoles without a change in dense bodies. A morphometric analysis of the content of the autophagic vacuoles showed that the endoplasmic reticulum membranes were preferentially removed, and from this we conclude that the formation of autophagic vacuoles was not a random process. Our findings show that the removal of excess cytoplasmic membranes is associated with an increase in autophagic activity and thus demonstrates the presence of a specific cellular mechanism which may be responsible for the bulk removal of PB-induced membranes.     

Degeneration and regeneration in denervated tonic and phasic skeletal muscle: Morphology and acid phosphatase cytochemistry

Following denervation, ultrastructural alterations were observed in the tonic, anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken. In the ALD muscle these changes were characteristic of both degeneration and regeneration, while in the PLD muscle, the changes were characteristic only of degeneration. Acid phosphatase positive structures, which included dense bodies in the ALD and PLD as well as T-tubules in the PLD, were observed intact with no evidence of release of enzyme into the sarcoplasm. No evidence of an increase in the number of autophagic vacuoles was found. The morphological evidence presented in this communication does not support the hypothesis that lysosomes are involved in denervation atrophy through autophagy of muscle cell constituents.     

Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.