Effects of glucagon on hepatic glycogen and smooth endoplasmic reticulum

The action of glucagon on hepatic glycogen and smooth endoplasmic reticulum (SER) was studied in samples of liver taken sequentially from anesthetized rats. The physiological state of each animal was assessed by continuously monitoring aortic blood pressure and blood lactate/pyruvate ratios. High hepatic glycogen levels were established by using 10–12 hr fasted control-fed rats infused continuously with glucose. In rats receiving glucose only, hepatic glycogen levels remained above 5.0% during the 4-hr period of glucose administration. Centrilobular hepatocytes displayed an abundance of glycogen which often appeared dispersed with elements of SER between the glycogen particles. Periportal cells had dense clumps of glycogen with few vesicles of SER restricted to the periphery of the glycogen masses. The addition of glucagon to the glucose infusate caused a marked stimulation of glycogenolysis. In these rats, the hepatic glycogen level (x?±SE) was 6.71±.15% 1 hr after glucose and declined after initiation of glucagon infusion as follows: 5.86±.29% (15 min), 4.89±.26% (1 hr), 2.16±.40% (2 hr), and 1.66±.29% (3 hr). The fine structure of hepatocytes showed a dramatic response to the administration of glucagon. The glycogen regions of the cells were noticeably decreased in size and number of glycogen granules 3 hr after initiation of glucagon infusion, and SER was abundant in both periportal and centrilobular hepatocytes. The interpretation offered is that glucagon induces the formation of new SER membranes which participate in glycogen breakdown and/or glucose release from hepatocytes.     

The effects of insulin replacement and withdrawal on hepatic ultrastructure and biochemistry

This study examines the early hepatic biochemical and ultra-structural responses to insulin replacement in streptozotocin-diabetic rats and insulin withdrawal from insulin-maintained diabetic rats. Insulin administration rapidly lowered plasma glucose and the elevated glucose-6-phosphatase (G-6-Pase) specific activity of the diabetic rats. However, hepatic glycogen did not increase until after 3 hr of insulin treatment. Hepatic ultrastructure responded to insulin replacement after the decline in glucose and G-6-Pase. This was seen in periportal hepatocytes as a reduction in the close association between smooth endoplasmic reticulum (SER) and glycogen particles in the diabetic animals. The treated rats showed hepatic SER restricted to the periphery of glycogen masses, as is characteristic of these cells from normal rats, in many cells by 6 hr and all cells by 18 hr. Insulin withdrawal from insulin-treated diabetic rats elicited nearly a total reversal of the above events. Plasma insulin declined to a value half that of the normal rats by 6 hr after withdrawal; concurrently, plasma glucose rose sharply to hyperglycemic values as hepatic glycogen content dropped. Following the rise in plasma glucose and fall in glycogen content, G-6-Pase specific activity increased and by 16 hr reached the high values characteristic of the diabetic animal. Hepatic ultra-structure was also changed as evidenced by an intrusion of elements of the SER into the dense glycogen masses; the result was dispersed glycogen closely associated with SER as seen in the diabetic animal. It is concluded that the hepatic response to insulin replacement in diabetic animals and diabetic onset in insulin-withdrawn animals is rapid and occurs through defined stages.     

Fine structure of hepatocytes from fasted and fed rats

The fine structure of hepatocytes from rats maintained on a controlled feeding schedule are described. Liver samples were processed for electron microscopy, histochemistry and chemical determinations of glycogen at precise time-intervals following a 30-hour fast and a 2-hour meal. Hepatocytes from 30-hour-fasted rats with extremely low hepatic glycogen levels were devoid of glycogen particles. Centrilobular cells showed areas of the cytoplasm rich in vesicles of smooth endoplasmic reticulum (SER) while periportal hepatocytes contained less extensive regions of SER. Soon after feeding the fasted rats, glycogen particles appeared in regions of the cell rich in SER. Centrilobular hepatocytes contained numerous glycogen areas which were infiltrated with tubules of SER, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the masses of glycogen. Throughout glycogen deposition each glycogen particle was closely associated with membranes of SER until maximum glycogen deposition was achieved 12 hours after initiation of feeding. At this point SER was reduced to the lowest amounts of the time-periods studied. During stages of glycogen depletion SER proliferated and reached the highest concentration measured in this study. Tubules of SER were present throughout the glycogen masses of centrilobular hepatocytes, whereas in periportal cells the organelle was restricted to the periphery of the glycogen masses. It is concluded that SER is associated with glycogen particles in rat hepatocytes during both deposition and depletion of glycogen.     

Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of 3H-galactose

Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting ³H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.     

Electron microscopical investigation on aldrin-induced hepatocyte pathology in Rana catesbeiana, with special emphasis on peroxisomes.

We examined the effect of aldrin on hepatocyte ultrastructure in liver of Rana catesbeiana. The frogs were experimentally exposed to chemical substance and liver fragments processed for routine transmission electron microscopy. Hepatic peroxisomes were visualized after incubation with alkaline 3,3'-diaminobezidine (DAB) method. Ultrastructural analysis revealed progressive hepatocyte changes induced by this drug. After 2-weeks, in the hepatocytes the nuclear envelop and the cisternae of both smooth and rough endoplasmic reticulum (SER und RER, respectively) were unusually enlarged. Reduction of glycogen granules associated with an increased frequency of lysosomes was observed. Normal appearing peroxisomes were present in clusters. Lipid droplets were also visualuzed. After 4-weeks, there was a new increase of glcogen associated with a great number of mitochondria and peroxisomes. Moreover, SER und RER were still dilated. Intracellular lipid inclusions became more abundant. These results suggest that the aldrin 250 induces ultrastructural changes in the hepatocyte of Rana catesbeiana.     

Action of metabolic hormones on the fine structure of rat liver cells III. Effects of adrenalectomy and administration of cortisone

Electron microscopic studies were made of hepatocytes from sham-operated rats, adrenalectomized animals fasted 15 hours, and adrenalectomized rats fasted 15 hours but given a single I.P. injection (10 mg) of cortisone acetate. The objective of this work was to define the earliest morphological response of hepatocytes to injection of a glucocorticoid and to provide additional information on the mechanism of hormone action at the cellular level. Hepatocytes from fasted, adrenalectomized rats contained no glycogen particles and very little smooth endoplasmic reticulum (SER). In addition the rough endoplasmic reticulum was disorganized and showed fewer ribosomes and polysomes than found in liver cells from sham-operated rats. Two hours after glucocorticoid injection glycogen particles were seen in numerous centrilobular cells and some periportal hepatocytes. Elements of SER were associated with the glycogen particles. By 4 hours after hormone injection abundant glycogen was found in all hepatocytes. Centrilobular cells showed dispersed glycogen with extensive tubules of SER associated with the glycogen particles. Periportal hepatocytes accumulated glycogen as dense masses scattered throughout the cytosome. SER occurred mainly at the edges of the glycogen masses. Midlobular cells showed glycogen patterns intermediate between periportal and centrilobular cells; masses of dispersed glycogen with abundant SER occurred within and around the glycogen areas of the cells. Glucocorticoid stimulation also caused cisternae of RER to align in parallel arrays, and more ribosomes and polysomes appeared on membranes of RER than in similar cells from adrenalectomized rats. The interpretation is offered that the glucocorticoid-stimulated proliferation of SER is the morphological expression of induced microsomal enzyme synthesis (glucose-6-phosphatase) known to occur under these hormonal conditions.     

Intracellular changes in rat hepatocytes after intratracheal administration of highly dispersed silicon dioxide and uridine effects on these changes

Rat hepatocytes were examined under electron microscope at early terms after intratracheal administration of highly dispersed silicon dioxide powder against the background of uridine treatment. Penetration of powder particles into hepatocyte cytoplasm, nuclei, mitochondria, and peroxisomes and development of bacteria in these cells were observed. Uridine reduced the destructive effect of powder on the organelles, increased glycogen content in hepatocytes, and inhibited the formation of capsulated bacterial forms in these cells. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 596–600, May, 2006     

Acute toxic effects of paraquat on ultrastructure of rat liver

Paraquat was administrated to pathogen-free male rats orally, and the livers were studied by light and electron microscope at intervals of 6 hours to 5 days. Congestion and hepatocellular injury (degeneration and/or fatty metamorphosis) were seen by light microscope. Electron microscope showed that degranulation of RER, proliferation of SER, decreasing of glycogen particles and mitochondrial swelling occurred in the cytoplasm of the hepatocytes within 2 layers around the central vein at 6 hours. After 12 hours the liver cells throughout the centrolobular area were injured. Degranulation of RER, proliferation of SER, and decreasing of glycogen particles became prominent, and mitochondria showed swelling and transformation. In the midzonal and periportal areas, numerous lipid droplets were seen in the cytoplasm of the hepatocytes. From the result of ultrastructural findings, it is considered that detoxication and biotransformation of paraquat occur in the hepatocytes within 2 layers around the central vein at an early stage, and spread to the hepatocytes throughout centrolobular area later.     

Alterations in hepatic fine structure after chronic exposure of rats to dexamethasone

The objective of this study was to determine the effects of chronic dexamethasone (DEX) administration on hepatic ultrastructure and to correlate these changes with plasma lipoprotein levels. Electron microscopic studies were made of hepatocytes from male rats killed 1, 3 and 5 days after DEX (2 mg, twice per day) administration. Three days after treatment plasma lipoprotein levels were highest and hepatocytes contained regions of the cytosome rich in elements of the smooth endoplasmic reticulum (SER). Osmiophilic particles were present in the tubules and vesicles of the SER, in the saccules and vacuoles of the Golgi complex, in secretory vesicles near the cell surface and in the space of Disse. DEX treatments also caused hepatocytes to accumulate tightly packed masses of β-particles of glycogen in some regions of the cell while other areas displayed dispersed glycogen particles that were associated with the SER. These observations are consistent with the hypothesis that glucocorticoids 1. cause an elevation of plasma lipoprotein levels by in creasing hepatic synthesis and secretion of VLDL, which involves the sequential participation of the ER, the Golgi complex and exocytosis of VLDL-containing vacuoles into the space of Disse, and 2. produce a change in the nature of the association of glycogen particles with the SER membranes in response to the physiological state of the animal.     

Alterations in hepatic fine structure after chronic exposure of rats to dexamethasone.

The objective of this study was to determine the effects of chronic dexamethasone (DEX) administration on hepatic ultrastructure and to correlate these changes with plasma lipoprotein levels. Electron microscopic studies were made of hepatocytes from male rats killed 1, 3 and 5 days after DEX (2 mg, twice per day) administration. Three days after treatment plasma lipoprotein levels were highest and hepatocytes contained regions of the cytosome rich in elements of the smooth endoplasmic reticulum (SER). Osmiophilic particles were present in the tubules and vesicles of the SER, in the saccules and vacuoles of the Golgi complex, in secretory vesicles near the cell surface and in the space of Disse. DEX treatments also caused hepatocytes to accumulate tightly packed masses of beta-particles of glycogen in some regions of the cell while other areas displayed dispersed glycogen particles that were associated with the SER. These observations are consistent with the hypothesis that glucocorticoids 1. cause an elevation of plasma lipoprotein levels by increasing hepatic synthesis and secretion of VLDL, which involves the sequential participation of the ER, the Golgi complex and exocytosis of VLDL-containing vacuoles into the space of Disse, and 2. produce a change in the nature of the association of glycogen particles with the SER membranes in response to the physiological state of the animal.     

Peroxisomes in the nervous system of Aplysia californica: a cytochemical study

Summary We have studied the distribution of peroxisomes in the abdominal ganglion ofAplysia californica using electron microscopic cytochemical methods. Reaction product for catalase was observed in small ovoid or dumb-bell-shaped bodies in the perikarya of many of the neurons. The abundance of these catalase-reactive peroxisomes is considerably greater than is the case in vertebrate neurons. While the non-neuronal cells of the Aplysia abdominal ganglion do contain appreciable peroxisome populations, there were few peroxisomes in glial cytoplasm directly adjacent to the perikarya, again contrasting with vertebrate ganglia in which the satellite cells are a principal site of peroxisomes.Peroxisomes are present throughout the perikaryal cytoplasm. In the regions in which lipochrome granules abound, peroxisomes are frequently seen closely associated with these granules; glycogen is abundant nearby. The association of peroxisomes, lipochrome granules and glycogen is interesting in view of the propinquities of peroxisomes to lipid droplets and lipofuscin granules reported for non-neuronal vertebrate tissues, and in view of the growing evidence indicating that some of the roles of peroxisomes are in lipid metabolism and in gluconeogenesis. Some of the lipochrome granules themselves show reaction product in ganglia incubated to demonstrate catalase activity and some react in tissue incubated to demonstrate acid phosphatase activity. Such observations suggest that the enzymatic capacities of the lipochrome granules merit further studies, and that the granules may be of complex or heterogeneous nature.     

Use of a monoclonal antibody against Lafora bodies for the immunocytochemical study of ground-glass inclusions in hepatocytes due to cyanamide

AIMS: Ground-glass inclusions (GGIs) in hepatocytes are known to be associated with cyanamide treatment in patients with alcohol dependency. The purpose of this study was to assess the reactivity of a monoclonal antibody (MAb) raised against polyglucosan and to detect early events in GGI formation. METHODS AND RESULTS: Formalin-fixed paraffin-embedded liver tissues from four patients treated with cyanamide were used. Sections were stained with haematoxylin and eosin and periodic acid-Schiff with and without diastase digestion, and were immunohistochemically stained with the MAb. For electron microscopic study, routinely processed liver tissue from one patient was examined with conventional and immunoelectron microscopy with use of the MAb. All specimens from the four cyanamide-treated patients contained GGIs in the cytoplasm of hepatocytes, and these GGIs reacted intensely with the MAb. Fully developed GGIs contained various organelles, whereas early ones consisted primarily of glycogen granules and dilated smooth endoplasmic reticulum. In immunoelectron microscopic preparations, gold particles were located within GGIs, and the immunolabelled organelles appeared to be glycogen granules. CONCLUSIONS: This novel MAb is useful for the detection of GGIs caused by cyanamide. Our results support the idea that GGI formation may result from specific abnormalities in glucose metabolism.     

Rapid alterations induced by insulin in hepatocyte ultrastructure and glycogen levels

The speed with which insulin alters hepatocyte ultrastructure and glycogen levels in insulin-deficient rats has been studied. Insulin deficiency was induced with alloxan, followed by insulin treatment with regular and NPH insulin. Rats were killed at various times after the insulin injection, blood samples were obtained, plasma glucose levels were determined, and liver samples were prepared for electron microscopy and glycogen determinations. Plasma glucose levels in insulin-deficient rats declined to normal values by 4 hours post insulin, returning to insulin-deficient levels by 8 hours post insulin. Hepatic glycogen was considerably reduced in the insulin-deficient rats. By 1 hour post insulin hepatic glycogen increased, reached maximal levels by 8 hours, then declined to insulin-deficient levels by 36 hours. The ultrastructural appearance of both centrilobular and periportal hepatocytes from insulin-deficient rats showed abundant vesicular smooth endoplasmic reticulum (SER), decreased rough endoplasmic reticulum (RER), and enlarged RER intracisternal spaces. One-half hour post insulin, centrilobular hepatocytes were unchanged. In periportal hepatocytes, however, vesicular SER was no longer visible, the RER intracisternal spaces appeared normal, and the amount of RER had increased. By 1 hour post insulin the centrilobular hepatocytes showed similar ultrastructural changes. These changes became more pronounced in the next few hours and remained through 24 hours. By 36 hours both centrilobular and periportal hepatocytes appeared similar to those in the insulin-deficient rat. These results demonstrate the rapid and lobular-specific effects insulin has on the hepatocyte.     

重型病毒性肝炎肝细胞“水样变性”性质的探讨

Specimens were fixed with a mixed fluid of osmium acid potassium ferrocyanide, and embedded with Epon--812. Totally, biopsies from 21 cases of severe chronic viral hepatitis were investigated. When hydropic degeneration of hepatocytes occurred, the clear field in cytoplasm were actually places for accumulating massive glycogen. The granules around the nuclei were convergent mitochondria, endoplasm reticulum and other organelles. Glycogen was best shown in this experiment because of adoption of the mixed fluid as the fixative, which successfully prevented the loss of glycogen during dehydration and the process of uranyl acetate staining. In our opinion this change in hepatocytes might better be termed "glycogen degeneration". It is probably related to infusion of large amount of glucose fluid or due to damage of certain glycogenolytic enzymes in the liver.     

Ca2+ and electrolyte mobilization following agonist application to the pancreatic β cell line HIT

We have investigated intracellular Ca2+ mobilization in oscillations of cytoplasmic Ca2+ in response to glucagon-like peptide 1 (GLP-1) and glucose in clonal HIT insulinoma cells with a confocal laser-scanning microscope (CLSM). We also used electron probe X-ray microanalysis to determine the GLP-1- and glucose-induced changes in electrolyte levels in the cytoplasm and insulin granules of the cells. GLP-1 produced 10- to 35-s duration oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i), both with and without Ca2+ in the extracellular solution, suggesting that Ca2+ is mobilized from intracellular Ca2+ stores, namely secretory granules. Glucose caused 1- to 3-min duration oscillatory increases in [Ca2+]i when the extracellular solution contained Ca2+. When the cells were cultured without Ca2+ (no Ca2+ added, 1 mM EGTA), an oscillatory [Ca2+]i increase of amplitude and short duration (12-35 s) was produced by 11 mM glucose, and the oscillation was inhibited by ruthenium red. X-ray microanalysis showed that stimulation with glucose increased the total Ca concentration in the cytoplasm and decreased it in the insulin granules with and without Ca2+ in the extracellular solution. The application of glucose significantly decreased K, and increased Na and C1 in the cytoplasm when the extracellular solution contained Ca2+. Our result also suggests that the [Ca2+]i oscillation induced by glucose is involved in the release of Ca2+ from intracellular Ca2+ stores through the ryanodine receptor, which is blocked by ruthenium red, and/or through the inositol trisphosphate receptor that may be present in the membrane of insulin granules.     

Impairment of glucokinase translocation in cultured hepatocytes from OLETF and GK rats, animal models of type 2 diabetes

We examined sugar-induced translocation of glucokinase in cultured hepatocytes from Otsuka Long-Evans Tokushima Fatty and Goto-Kakizaki rats, animal models of type 2 diabetes, and compared this with that in Long-Evans Tokushima Otsuka and Wistar rats, respectively, as control strains. When hepatocytes from the four strains were incubated with 5 mM glucose, glucokinase was present predominantly in the nuclei. Higher concentrations of glucose, 5 mM glucose plus 1 mM fructose, and 5 mM glucose plus 1 mM sorbitol all induced the translocation of glucokinase from the nucleus to the cytoplasm in hepatocytes from these rats. The extent of glucokinase translocation under these conditions, however, was less marked in both diabetic rat types than in the control rats. The extent of the phosphorylation of glucose as estimated by the release of 3H2O from [2- 3H] glucose is significantly lower in Goto-Kakizaki rats than in Wistar rats. The results indicate that the translocation of glucokinase is impaired in the hepatocytes of diabetic rats. They also suggest that the impaired translocation of glucokinase is associated with abnormal hepatic glucose metabolism in type 2 diabetes.     

Localization of glycogen synthase activity in liver of fasted normal and adrenalectomized rats after injection of dexamethasone

Hepatic glycogen synthase activity was localized in normal and adrenalectomized (ADX) rats after fasting overnight and in fasted ADX rats after injection of dexamethasone (DEX) 2–8 h prior to sacrifice to stimulate glycogen synthesis. Cryostat sections were incubated in medium containing substrate to demonstrate glycogen synthase activity as indicated by glycogen synthesized during incubation. Sections from fasted normal rats showed limited dispersed glycogen synthase activity in both periportal and centrilobular regions. In contrast, activity for glycogen synthase in hepatocytes from fasted ADX rats appeared as large aggregates in random hepatocytes throughtout the lobule. Two hours after injection of DEX the reaction product appeared as aggregates in some hepatocytes, but other cells revealed dispersed enzyme activity. Glycogen synthase activity was evident in more hepatocytes after 4 h treatment with DEX and after 8 h virtually all hepatocytes contained abundant reaction product. The results suggest that synthase activity becomes concentrated in limited regions of selected hepatocytes in fasted ADX rats. DEX stimulation of glycogen synthesis for 4–8 h results in increased enzyme activity. © 1993 Wiley-Liss, Inc.     

FINE STRUCTURAL COMPARISON OF EWINGS SARCOMA WITH NEUROBLASTOMA

Two cases of Ewing's sarcoma and two of neuroblastoma, rosette forming and round cell type, were studied electron microscopically and their fine structures were compared. The neoplastic cells of Ewing's sarcoma were characterized by aggregated glycogen particles in the cytoplasm. They had pseudopod-like cytoplasmic processes having tight junctions, which never contained microtubules or mitochondria. Ewing's sarcoma cells exhibited several stages of cell maturation and some mature cells possessed a large amount of smooth and rough endoplasmic reticulum, large Golgi complexes and numerous phagosomes containing glycogen particles as well as cytoplasmic organelles. The neoplastic cells of neuroblastoma, rosette forming type, were characterized by synaptic junctions and numerous cytoplasmic processes with production of neurites containing microtubules, neurofibrils, mitochondria and a few catecholamine granules. A few cytoplasmic processes containing mitochondria were observed even in the round cell type.     

Fine structural comparison of Ewing's sarcoma with neuroblastoma.

Two cases of Ewing's sarcoma and two neuroblastoma, rosette forming and round cell type, were studied electron microscopically and their fine structures were compared. The neoplastic cells of Ewing's sarcoma were characterized by aggregated glycogen particles in the cytoplasm. They had pseudopod-like cytoplasmic processes having tight junctions, which never contained microtubules or mitochondria. Ewing's sarcoma cells exhibited several stages of cell maturation and some mature cells possessed a large amount of smooth and rough endoplasmic reticulum, large Golgi complexes and numerous phagosomes containing glycogen particles as well as cytoplasmic organelles. The neoplastic cells of neuroblastoma, rosette forming type, were characterized by synaptic junctions and numerous cytoplasmic processes with production of neurites containing microtubules, neurofibrils, mitochondria and a few catecholamine granules. A few cytoplasmic processes containing mitochondria were observed even in the round cell type.     

Clear cell chondrosarcoma: an ultrastructural study

Clear cell chondrosarcoma (CCC) is a rare neoplasm. We report here a case of CCC. A 67-year-old Japanese man presented with right arthralgia for 1 year, and histological examination of the subsequent surgical resection of the right femoral bone showed the finding of CCC. Ultrastructurally, most organelles were observed in the perinuclear area. Clear neoplastic cells contained many glycogen particles in the area of the cytoplasm lacking organelles, although glycogen particles overall seemed to be evenly distributed in the cytoplasm. Some mitochondria, Golgi complex, actin-like filaments, and rough endoplasmic reticulum were also demonstrated in the cytoplasm of clear cells. Well-developed microvilli were also seen on the surface of neoplastic cells. These structures in neoplastic cells corresponded notably to structures of normal chondrocytes. Finally, our ultrastructural findings support further evidence that clear cells in CCC may show chondrocyte differentiation and a lack of an organelles area as well as abundant glycogen particles, may contribute to the clear cell morphology in CCC.