Follicular Lumen Research Articles

Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland.

A sugar-binding protein, or endogenous lectin, was localized on fixed and paraffin-embedded thyroid sections by means of fluorescein-labelled neoglycoproteins. Fluorescence microscopy showed the binding of this lectin to be dependent on calcium ions and acidic pH and indicated sugar specificity for GlcNAc moieties only. In human, porcine and murine thyrocytes, specific binding was observed mainly on subcellular organelles. Conversely, in rabbit thyrocytes, specific labelling was seen mostly at the apical cell surface facing the follicular lumen. The possibility that this novel endogenous lectin is involved in the Tg metabolism is considered.

The N-acetylglucosamine-specific receptor of the thyroid. Binding characteristics, partial characterization, and potential role.

The binding characteristics of the GlcNAc binding protein present in thyroid membranes (Consiglio, E., Shifrin, S., Yavin, Z., Ambesi-Impiombato, F.S., Rall, J.E., Salvatore, G., and Kohn, L.D. (1981) J. Biol. Chem. 256, 10592-10599) were reinvestigated using neoglycoproteins as probes. Plasma membrane preparations from porcine thyroid specifically bound 125I-GlcNAc35-bovine serum albumin. Binding was dependent on the presence of calcium. Binding of ligand to receptor was minimal at neutral pH and maximal at pH 5.0. Equilibrium binding studies indicated positive cooperativity of binding and a site capacity of about 60 pmol/mg of protein. Competition studies were compatible with a specificity hierarchy of GlcNAc much greater than Gal; no recognition of mannose, fucose, or glucose moieties was noted. The receptor was detergent-solubilized from plasma membrane preparations and on the basis of the defined binding properties, purified by chromatography on a GlcNAc-Sepharose affinity column. The purified GlcNAc thyroid receptor has a subunit molecular size of about 45 kDa and appears to be an oligomer composed of three subunits. The receptor was identified as a component of thyrocytes by in situ cytochemical localization with fluorescent neoglycoproteins. In certain cases it was mainly present on, or near, the apical cell surface. It is suggested that this GlcNAc receptor functions in thyroglobulin metabolism, possibly involved in recycling of internalized thyroglobulin molecules back into the follicular lumen.

TSH and biogenic amine signals in the regulation of thyroid function: independent regulation by protein kinase C and G proteins.

The primary role of the thyroid cell is to synthesize and secrete the thyroid hormones, T3 and T4 (Dumont et al. 1971; Robbins et al. 1980). This must be accomplished in a regulated manner and despite a variable intake of iodine. This role must also be accomplished despite the fact that the synthesis and secretion of thyroid hormones involves numerous steps. These include 1) the uptake of iodide across the basal membrane of the cell in a scavanging and concentrative fashion; 2) efflux of iodide across the apical membrane into the follicular lumen of the thyroid follicle; 3) the synthesis of thyroglobulin and its vectorial transport to the site of iodide efflux; 4) iodination of thyroglobulin; 5) regulated storage of the iodinated thyroglobulin; 6) lysosomal targeting and biodegradation, of the iodinated thyroglobulin; and 7) ultimately, secretion of T3/T4 into the bloodstream, across the basal membrane

Norepinephrine and thyrotropin stimulation of iodide efflux in FRTL-5 thyroid cells involves metabolites of arachidonic acid and is associated with the iodination of thyroglobulin.

Ca2+-dependent and TSH-, norepinephrine (NE)-, and A23187-induced iodide (I-) efflux from FRTL-5 rat thyroid cells is inhibited by quinacrine and trifluoroperazine, agents that inhibit phospholipase A2 activity. Furthermore, I- efflux can be stimulated by an activator of phospholipase A2 activity, melittin. Phospholipase A2 action releases arachidonic acid from phospholipids; arachidonic acid enhances I- efflux in FRTL-5 cells. Inhibitors of arachidonic acid metabolism via the lipoxygenase pathway, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, and via the cytochrome P450-linked epoxygenase pathway, piperonyl butoxide and 2-diethylaminoethyl-2,2-diphenyl valerate, but not an inhibitor of the cyclooxygenase pathway, indomethacin, can inhibit TSH-, NE-, and A23187-induced I- efflux. TSH, NE, and arachidonic acid stimulation of I- efflux in FRTL-5 cells is associated with increased iodination of thyroglobulin, which is blocked by 10 microM 5,8,11,14-eicosatetraynoic acid and 50 microM piperonyl butoxide. The data thus suggest that TSH- and NE-induced I- efflux from FRTL-5 thyroid cells involves lipoxygenase and/or epoxygenase metabolites of arachidonic acid, released from phospholipids upon Ca2+-dependent activation of phospholipase A2. Since this process is associated with the iodination of thyroglobulin, TSH- and NE-induced I- efflux in FRTL-5 cells may represent the transport of I- from the cell into the follicular lumen in vivo.

Phorbol myristate acetate inhibits alpha 1-adrenergically but not thyrotropin-regulated functions in FRTL-5 rat thyroid cells.

The iodination of thyroglobulin and the formation of thyroid hormones are regulated by alpha 1-adrenergic agents as well as TSH in rat FRTL-5 cells. The regulatory effects of the alpha-1-adrenergic agents and TSH on both of these processes are associated with an increase in cytosolic Ca2+ and an increase in that component of iodide efflux that is representative of the movement of iodide from the thyroid cell into the follicular lumen. When FRTL-5 cells are preincubated with phorbol myristate acetate (PMA) for at least 3 min, the norepinephrine-stimulated changes in cytosolic Ca2+ levels and iodide efflux are inhibited. In contrast, PMA pretreatment has no effect on iodide efflux and actually enhances the changes in cytosolic Ca2+ induced by TSH. Phorbol myristate acetate pretreatment has no effect on TSH-stimulated cAMP-mediated iodide uptake in FRTL-5 cells, nor does it affect the binding parameters of the alpha 1-adrenergic receptor antagonist prazosin. These data suggest that protein kinase C is involved in a feedback mechanism regulating alpha 1-adrenergic but not TSH-induced changes associated with the iodination of thyroglobulin and the formation of thyroid hormones; and that this feedback effect occurs after the step of ligand binding but before the increase in cytosolic Ca2+ induced by the alpha 1-adrenergic agents.

Epithelial cell polarization in culture: orientation of cell polarity and expression of specific functions, studied with cultured thyroid cells.

Isolated porcine thyroid cells reorganize in culture into various types of multicellular structure, which differ in the orientation of cell polarity and in the surface of the cell layer accessible to molecules present in the culture medium. The types of structure are: (1) follicles: the basal pole is oriented toward the medium; (2) inside-out follicles or monolayers: the apical pole is facing the culture medium; (3) monolayers on a permeable substratum: both sides of the cell layer are accessible to the medium. Follicles can be transformed into inside-out follicles or monolayers and vice versa by manipulation of the external cell environment and without dissociating the cells. Cells concentrate iodide and respond to acute stimulation by thyroid-stimulating hormone (TSH) when the basal pole is accessible, and organification occurs only when cells form a closed follicular lumen. In porous-bottomed culture chambers monolayers are formed with the basal surface accessible to the medium and the apical compartment separated from the medium. Under these conditions 85-95% of the thyroglobulin produced is secreted apically and 5-15% basally. Thyrotropin stimulates (X3) apical accumulation without modifying secretion in the basal compartment. Sodium transport across the cell layer has been characterized. An amiloride-sensitive influx occurs at the apical pole whereas the Na+/K+-ATPase, localized in the basolateral membrane, mediates ouabain-sensitive efflux at the basal pole. The thyroid epithelium in culture appears therefore as a Na+-absorbing epithelium. The role of this transport in the stabilization of cell polarity is discussed.

Mixed follicles of the human thyroid gland.

Mixed follicles are structures composed of squamous-like and follicular epithelia. Little attention has been generally paid to these peculiar follicles of the human thyroid; thus the aim of the present study was to investigate their prevalence and biological properties by means of systematic autopsy, histochemical and immunohistochemical surveys. Mixed follicles were found to be present in 54% and 81% of the patients with solid cell nests, as well as in 50% and 77% of the total number of the ultimobranchial nests, when one or two histological samples from each solid cell nest were examined, respectively. The follicular lumen of mixed follicles usually contained an eosinophilic and PAS-positive colloid-like material, although in 22% of the cases acid mucins sometimes intermixed with PAS-positive granular material and cell debris were also present within lumina. Follicular cells lining mixed follicles basically did not stain positively for calcitonin. The results indicate that mixed follicles are not rarely found in the human thyroid. The presence of intraluminal mucins and cell debris and the absence of calcitonin-containing cells in the follicular epithelium lining these peculiar follicles suggest that at least some thyroid follicular cells could originate from ultimobranchial tissue.

Effects of hydrocortisone on the ultrastructure of the thymic cysts of chicks

Thymi from 10-day old male chicks injected subcutaneously with 5 mg of hydrocortisone for 2 consecutive days and sacrificed on the 4th day were examined by electron microscopy. With depletion of the lymphoid cells, cystic structures, consisting of inter- and intracellular cysts are more apparent in the hydrocortisone-treated chicks. The intercellular cysts vary in size and shape and are lined by cystic, endocrine-like and lymphoid cells. Luminal surfaces of the cystic cells contain microvilli. The lateral plasma membranes show complex interdigitations. The prominent terminal web contains vesicles and numerous core rootlets. Frequent membrane invaginations are apparent at the bases of the microvilli. The endoplasmic reticulum and the Golgi complex are moderately developed. Mitochondria are variable in number but are numerous in some cells. The cytoplasm also contains dense bodies, vacuoles and myelin figures. Endocrine-like cells, containing numerous secretory granules and bordering directly on the follicular lumen suggest that their secretion may be discharged into the lumen. Cystic cells containing intracellular cysts are observed. Presence of cleft-like spaces, microvilli closely opposed to adjacent cytoplasmic processes or lateral microvilli may reflect responses to increase in absorptive or transport function. The present data suggest that hydrocortisone may modulate the structure and function of the cystic structures in the chick thymus.

Effect of isotretinoin on experimentally induced comedones.

The effect of isotretinoin on experimentally induced comedones was studied by means of histoplanimetry and scanning electron microscopy (SEM). To induce comedone formation, insoluble cutting oil was applied to the ventral surface of the ears of rabbits for 2 weeks. After comedones formed, isotretinoin was fed to the rabbits by a feeding tube, daily, for 4 weeks. A low dose (2.0 mg/kg) and a high dose (20.0 mg/kg body weight) were selected for two different groups. Soybean oil served as the vehicle. Histoplanimetrically, the high-dose group showed a significant decrease in size of comedones when compared to the control group and the low-dose group [P less than 0.005], and in the high-dose group, the follicular lumen contained a small number of loose, non-adherent, horny cells. Upon SEM examination, the comedones were seen to have a 'chrysanthemum' appearance before treatment. After treatment, the high- and low-dose groups both showed evidence of the elimination of comedones on three-dimensional analysis.

Autoimmune-resistance in Graves' disease tissues indication of a structural and functional heterogenicity

Out of 256 patients with Graves' disease and a characteristically low echogenic ultrasound appearance of the thyroid gland, 12 patients (4.7%) showed additionally one single or several intrathyroidal focal alterations with high echogenicity. Their volume was 0.2 to 7.0 cm (X +/- SD: 1.75 +/- 2.24 cm). A common characteristic of the 12 patients was a longstanding history of Graves' disease and antithyroid treatment with thiamazol or carbimazol and a mean age of 55 +/- 9 (SD) years (range 42-75 years). The foci with high echogenicity had a normal or macrofollicular histological structure (mean follicle lumen diameter 119.7 micron, as proven by histology in 4 cases) which was significantly higher than follicle lumina of the surrounding Graves' disease tissue (mean follicular lumen diameter 38.3 micron; P greater than 0.001). The height of follicle epithelium and the nuclei cross-section area of such foci was similar to corresponding normal tissue parameter in 5 out of 7 foci. The 99mTc-uptake of the foci amounted to 40 to 80% in comparison with Graves' disease tissue. 131-iodine autoradiography, thyroglobulin immunochemistry and 125-iodine-TSH binding analysis performed in selected cases confirmed the heterogenicity of these foci. The data provided evidence that the foci were unresponsive to TSH-receptor mediated stimulation through Graves' immunoglobulins. It is concluded that longstanding Graves' disease can be associated with tissue foci which are in a functional sense resistant to immunogenic stimulation by TSH-receptor autoantibodies.

Thyrotropin-induced formation of functional follicles in primary cultures of ovine thyroid cells

In primary cultures of ovine thyroid cells, TSH induced the expression of several differentiated functions including the formation of follicles, and synthesis and storage of iodinated thyroglobulin in the follicular lumen. In the present report, these follicles were shown by transmission (TEM) and scanning electron microscopy (SEM) to be intact, comprised of two or more cells and to possess numerous microvilli on the inner cell membranes facing the follicular lumen. The TSH-induced formation of follicles was reversible and dynamic, with the kinetics of formation preceding that of iodination. The follicles were further demonstrated to be functional in terms of thyroglobulin storage and iodination.

Serum Thyroglobulin and Thyroid Cancer

Thyroglobulin is a large glycosylated protein (MW 660000 daltons) which is synthesised and secreted by the thyroid cell into the follicular lumen. In the thyroglobulin molecule the iodotyrosines undergo oxidative coupling to form the iodothyronines thyroxine (T4) and triiodothyronine (T3). Production of thyroid hormones results from micropinocytosis of iodinated thyroglobulin from the colloid into the thyroid cell and its hydrolysis by lysosymal enzymes. The resulting T4 and T3 diffuse across the basal membrane into the blood stream whilst iodotyrosines are de-iodinated and the iodine recycled. Thyroglobulin thus acts as the template for thyroid hormone synthesis and a storage form of inactive thyroid hormones and iodine. Thyroid function and activity are under the positive control of thyrotrophin (TSH) which stimulates many steps of thyroid cell metabolism including protein synthesis, iodothyronine synthesis, thyroglobulin secretion into the follicular lumen, and replacement of micropinocytosis of thyroglobulin by massive colloid macropinocytosis [1]. The action of TSH on the thyroid cell is mediated via adenylate cyclase activation and recent work has shown that transcription of the thyroglobulin gene is regulated by cyclic AMP [2]. For many years thyroglobulin was thought to be confined to the thyroid gland but the sensitive radioimmunoassays now available have demonstrated that the protein is unequivocally present in the peripheral circulation of man. It is assumed that some of the protein escapes the potent lysosymal mechanism of the thyroid gland and crosses the basal membrane with the thyroid hormones to appear intact in the circulation. The first radioimmunoassays for thyroglobulin in human, monkey and rat serum were reported in 1967 [3]. A wide variation in the normal range of human serum thyroglobulin levels has been reported but a recent international co-operative study evaluating serum thyroglobulin standards has shown this to be due to remarkable interlaboratory variations [4]. No age-related variation of thyroglobulin levels has been reported (except in children) although several studies have reported higher values in women than men, higher values in pregnant women at delivery than non-pregnant controls, and elevated thyroglobulin levels in cord blood from newborn infants. Serum concentrations in man appear to be closely regulated within a narrow range suggesting a mechanism for controlling thyroglobulin release. Thyroid hormone administration suppresses thyroglobulin levels and after injection of thyrotrophin releasing hormone, TSH or thyroid stimulating immunoglobulins, the levels of serum thyroglobulin rise. The data support the concept that thyroglobulin is a normal secretory product of the thyroid gland under the control of TSH, but the mechanism of this 'secretion1, rather than simple diffusion, remains unknown [3].

Fluctuations in follicular structures of rat thyroid glands during 24 hours: Fine structural and morphometric studies

The thyroid follicles of adult male Wistar rats were examined at six evenly spaced times over 24 hr with a morphometric technique. Follicular structures were subjected to distinct variations during 24 hr, with respect to volume and numerical densities of follicles in the thyroid gland, and diameters, volumes, cell numbers, and luminal surface areas of individual follicles. Variations in follicular structures were divided into two phases: a large follicular phase at 1200, 1600, and 2000 hr and a small follicular phase at the other times. Although volume densities of follicles in the gland varied with a small amplitude, diameters, volumes, and cell numbers of individual follicles exhibited distinct fluctuations during 24 hr. Numerical densities of follicles in the gland changed distinctly during the small follicular phase as well. Degenerating follicular cells appeared in the follicular lumen especially at 1600 hr. No mitotic follicular cells were found throughout the experiment. Furthermore, one to three follicular cells of two adjacent follicles were often in contact with each other at 0400, 0800, and 1200 hr, and these follicles were lined by the common basement membranes. These results suggest that the variations in follicular structures during the small follicular phase occur in the form of follicle separation and fusion. Moreover, the morphological and morphometric variations in follicles reflect those in subcellular structures of follicular cells previously reported by us.

Ultrastructural modifications of the cavities formed by folliculo-stellate cells in chicken adenohypophysis under septic shock conditions.

Effects of septic shock by repeated inoculations with Escherichia coli on the ultrastructure of the folliculo-stellate cells and cavities of the adenohypophysis of the chicken were investigated in order to determine the function of these cavities. The principal morphological modifications were dilation of the Golgi apparatus, endoplasmic reticulum and autophagic vacuoles, and necrosis phenomena in the stellate cells. The follicular cavities showed dilation, and there was heterogeneous dense material and granular elements in the follicular lumen. Based on results reported in the literature, the observations reported here are evidence of a "cleaning-role", for the removal of cell debris, when there is endocrine disfunction.

Thyrotropin upregulates alpha 1-adrenergic receptors in rat FRTL-5 thyroid cells.

FRTL-5 rat thyroid cells grown and maintained in a medium containing 0.05 nM thyrotropin have a 10-fold higher number of alpha 1-adrenergic receptors on their cell surface than FRTL-5 cells maintained in the absence of thyrotropin in their medium. The increased number of alpha 1-adrenergic receptors, measured as increased specific [3H]prazosin binding per microgram of DNA, is not associated with any changes in Kd values for prazosin. Thyrotropin increases the number of alpha 1-adrenergic receptors by inducing their biosynthesis, as evidence by the inhibitory effects of cycloheximide or actinomycin D; the effect on biosynthesis is cAMP-mediated, since 8-bromoadenosine 3',5'-cyclic monophosphate, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine can mimic the thyrotropin effect in both extent and time course. The alpha 1-adrenergic receptors on FRTL-5 thyroid cells have been functionally linked to iodide efflux into the follicular lumen and to the iodination of thyroglobulin--i.e., to the formation of thyroid hormones; the alpha 1-adrenergic receptor signal is mediated by Ca2+ rather than by cAMP and involves arachidonic acid intermediates. The present data thus describe a unique upregulation phenomenon wherein the sequential expression of two receptors (thyrotropin and alpha 1-adrenergic) and two distinct signal systems (cAMP and Ca2+) are apparently a necessary prelude to thyroid hormone homoeostasis.

Honeycomb structure of the apical surface of thyroid epithelium during involution of the hyperplastic thyroid gland

A heretofore undescribed feature consisting of closely packed pits 0.5-1 μm in diameter was observed on the apical surface of thyroid epithelial cells. The walls of the pits were generally smooth, except at the base where there was a high incidence of irregularities looking like sites of recent fusion of apical vesicles with the pits. The matrix of the partition between pits was similar to the matrix of pseudopods in these cells in being free of membrane-bounded organelles and containing a low concentration of ribosomes. The pits were observed early in the involution of the hyperplastic gland, most prominently between 14 h and 8 days of involution. This is a time when thyroglobulin is accumulating in follicular lumens and the apical end of the epithelial cells usually contains a high concentration of apical vesicles ordinarily considered to be exocytic in character. It is important to recognize the existence of this feature to avoid confusing profiles of it with structures involved in macropinocytosis.

Fine structures of the thyroid gland in the aged central myelin deficient jimpy mouse.

The thyroid glands of 19-25 days old hypomyelinated jimpy mice were embedded in Araldite for morphological studies. The fine structures of the thyroid follicular epithelial cells were characterized by numerous microvilli, well developed rough endoplasmic reticulum with dilated cisternae, large and variously shaped mitochondria , groups of Golgi complex and abundant dense-cored vesides, bizzare shaped pseudopodos and many reabsorbed colloid droplets, and a large nucleus with abundant nuclear pore complex. Occasionally, cilia protruded from the apical surface of epithelial cells into the follicular lumen. In addition , mito-chondria-rich cells, clear cells (c-cells) , ergastoplasm-rich cells, and fenestrated intraepithelial capillaries were found on the follicular wall. Among thyroid foilicles, some incompletely differentiated epithelial masses appeared, which were composed of microfollicular epithelial cells, active mitotic epithelial cells , and small follides. These components were characteristic structures of the early embryonic development and represented the active proliferating stage of folliculogenesis. There were three types of cells observed on the wall of well de-veloped ultimobranchial cysts; i.e., secondary follide cells,c-cells , and ciliated cells. The presence of cysts implicated the underdevelopment of thyroid follicles which normally compressed and inhibited the persistence of ultimobranchial cysts . The evidence presented here proves that the hypomyelination of jimpy mice may be induced by the under - development of their thyroid glands and jimpy genes.

Thyroglobulin and calcitonin immunoreactivity in canine thyroid carcinomas.

Although thyroid gland neoplasms are well-recognized entities in dogs, the diagnosis and classification of these tumors often is difficult. In contrast to human thyroid carcinomas, which are predominantly of the papillary or follicular types, a relatively high proportion of the canine tumors contain compact cellular areas and resemble, to some extent, medullary thyroid carcinomas. In order to assess the value of immunohistochemical techniques in the identification and classification of these neoplasms, 21 canine thyroid carcinomas were examined for the presence of thyroglobulin and calcitonin using the peroxidase-antiperoxidase technique. Four major patterns of thyroglobulin immunoreactivity were present in the tumors, including diffuse cytoplasmic positive reaction, apical staining in the cells bordering the neoplastic follicular lumens, intracytoplasmic droplet staining, and staining of intrafollicular colloid. All follicular and mixed compact cellular/follicular tumors contained immunoreactive hormone, while only four of six compact cellular carcinomas were thyroglobulin-positive. The extent of thyroglobulin reactivity was consistently greater in tumors of the follicular and mixed patterns than in carcinomas of the purely compact cellular type. Two of four metastases, each of which retained the mixed pattern of the primary tumors, were thyroglobulin-positive. No medullary thyroid carcinomas were identified, but scattered calcitonin-positive cells in one mixed and in one compact cellular tumor were interpreted as entrapped nonneoplastic C cells. Immunohistochemical localization of thyroglobulin should facilitate the diagnosis of canine tumors of suspected thyroid follicular cell origin, particularly those arising in ectopic sites (i.e., heart base) and those presenting as metastases.

Endocytosis of human thyroglobulin (TG) in adenomatous goiter studied by an immuno-electron microscopic procedure.

Five cases of adenomatous goiter have been studied by an electron microscope using an immuno-reaction for thyroglobulin (TG) and focusing on the mechanism of endocytosis. Positive stain for TG was demonstrated in follicular lumina, large reabsorbed colloid droplets and small subapical vesicles. Endocytotic vesicles ranging from 320 nm to 1600 nm in diameter were observed in the cytoplasm as pits in the apical plasma membrane. Some of them showed direct connection with the positive stain for TG in the follicular lumen and the others were completely ingested in the cytoplasm. With statistic analysis, a majority of the vesicles showing the positive stain for TG in the cytoplasm distributed in the range of 200 nm to 1200 nm in diameter with the peak in 300 nm to 399 nm and was situated within an extent of the diameter measured from the endocytotic vesicles. Engulfment of colloid by pseudopods and fusion of the reabsorbed colloid droplets were encountered as extremely rare findings and appeared to play no major role for formation of large colloid droplets in adenomatous goiter.

Immunoelectron microscopic localization of thyroglobulin in human follicular adenoma.

An electron microscopic immunohistochemical localization of thyroglobulin (TG) using PAP methods has been made in 15 cases of cold follicular adenoma. All cases of follicular adenoma showed organ specific functions such as synthesis, storage, reabsorption, and hydrolysis of thyroglobulin except for an area composed of follicular cells with trabecular arrangement. Immuno-reaction product for TG was precisely demonstrated in follicular lumina, subapical vesicles and reabsorbed colloid droplets. The reaction product observed in the follicular lumen was clearly demarcated from the cytoplasm of the follicular cells by the apical plasma membrane. The subapical vesicles ranging approximately from 50 millimicrons to 300 millimicrons in diameter were rarely observed in follicular adenoma and some of them fused with the reabsorbed colloid droplets. The reabsorbed colloid droplets usually had the intense reaction product and hydrolyzed colloid droplets had a vacuole containing floccular low electron dense materials. There is no reaction product in rough endoplasmic reticulum and Golgi complexes.