Fine structural localization of peroxidase in the rat thyroid

Summary The fine structural localization of a peroxidase activity in the rat thyroid follicular epithelial cell was studied by histochemistry at electron microscopic level. The reaction product is recognized chiefly in the cisternae of the elements of granular endoplasmic reticulum and of nuclear envelope. Golgi vesicles or apical small vesicles, mitochondria, and dense granules are sometimes positive for this reaction. The relationship between the fine structural localization of peroxidase and the site of the iodination of thyroglobulin is discussed.     

APPEARANCE AND FUNCTION OF ENDOGENOUS PEROXIDASE IN FETAL RAT THYROID

Iodination within the thyroid follicle is intimately associated with a thyroid peroxidase. In order to locate the in vivo site of iodination, the initial cytochemical appearance of this enzyme has been determined in fetal rat thyroid and its presence correlated with the onset of iodinated thyroglobulin synthesis. Peroxidase first appears in follicular cells during the 18th day of gestation. It is seen first in the perinuclear cisternae, the cisternae of the endoplasmic reticulum, and within the inner few Golgi lamellae. These organelles presumably represent sites of peroxidase synthesis. During the 19th and 20th days of gestation, there is a tremendous increase in peroxidase activity. In addition to the stained sites described, there are now many peroxidase-positive apical vesicles in the follicular cells. Newly forming follicles stain most conspicuously for peroxidase, the reaction product being heavily concentrated at the external surfaces of apical microvilli and in the adjacent colloid. Iodinated thyroglobulin becomes biochemically detectable in thyroids during the 19th day of gestation and increases greatly during the 20th day. The parallel rise in peroxidase staining that just precedes, and overlaps, the rise in iodinated thyroglobulin, suggests that apical vesicles and the apical cell membrane are the major sites of iodination within the thyroid follicle.     

Endogeneous peroxidase activity in the frog thyroid gland

Summary The fine structural localization of the endogeneous peroxidase activity in the thyroid of the young frog was studied. The reaction product for peroxidase was observed over the peripheral luminal colloid and apical region of the follicular epithelial cell. Most apical small granules and some parts of Golgi lamellae and a few Golgi vesicles were specifically stained. The cisternae of rough endoplasmic reticulum and the nuclear cisternae did not demonstrate any positive reaction for peroxidase activity with difference from that of various cells of mammalia. In this study, only mature peroxidase seems to be positive for its reaction and the enzyme in the rough endoplasmic reticulum is considered to be too immature to react for DAB method in the frog thyroid cell. The relationship between the localization of peroxidase reaction and the site of the iodination of thyroglobulin was discussed.     

Intracellular trafficking of thyroid peroxidase to the cell surface

For thyroid hormone synthesis, thyroid peroxidase (TPO) molecules must be transported from the endoplasmic reticulum via the Golgi complex to be delivered at the cell surface to catalyze iodination of secreted thyroglobulin. Like other glycoproteins, TPO molecules in transit to the cell surface have the potential to acquire endoglycosidase H resistance as a consequence of Golgi-based modification of their N-linked carbohydrates, and measurement of the intracellular distribution of TPO has often relied on this assumption. To examine TPO surface distribution in thyrocyte cell lines, we prepared new antibodies against rat TPO. Antibody reactivity was first established upon expression of recombinant rat (r) TPO in 293 cells, which were heterogeneous for surface expression as determined by flow cytometry. By cell fractionation, surface rTPO fractionated distinctly from internal pools of TPO (that co-fractionate with calnexin), yet surface TPO molecules remained endoglycosidase H (endo H)-sensitive. Although the FRTL5 (and PC Cl3) rat thyrocyte cell line also exhibits almost no endo H-resistant TPO, much of the endogenous rTPO is localized to the cell surface by immunofluorescence. Similar results were obtained by fractionation of FRTL5 cell membranes on sucrose gradients. We conclude that in FRTL5 cells, a large fraction of rTPO is delivered to the plasma membrane yet does not acquire Golgi-type processing of its N-glycans. Rat and mouse thyroid tissue TPO also shows little or no endo H resistance, although cell fractionation still needs to be optimized for these tissues.     

MATURATION OF THE RAT FETAL THYROID

Maturation of the rat fetal thyroid was studied with the aid of I¹³¹ and of fluorescence and electron microscopy. The I¹³¹ concentration of the fetal gland increased exponentially from day 17 to day 20 of gestation and was related to the weight of the fetus (and presumably the weight of the thyroid) and also to the quantity of I¹³¹ accumulated by the fetus. In the 17-day gland, thyroglobulin or immunologically similar material was sparsely present in the incipient lumens of some cell clusters. With maturation, this material increased and was also observed within follicular cells on days 18 to 19 of gestation. On day 20, the specifically reacting material was present in the follicular lumens and was absent from the cytoplasm of follicular epithelium. Ultrastructurally, the earliest thyroid cells examined were replete with all the organelles found in the more mature epithelium. No direct correlation could be made between the cytoplasmic structures and the presence of thyroglobulin, although the granular endoplasmic reticulum was most likely the organelle responsible for synthesis of thyroglobulin. Thyroglobulin or a precursor was found in fetal thyroid cells before measurable quantities of I¹³¹ were concentrated and before cytoplasmic droplets appeared.     

Cytochemical localization of peroxidase and hydrogen-peroxide-producing NAD(P)H-oxidase in thyroid follicular cells of propylthiouracil-treated rats

The distribution of endogenous peroxidase and hydrogen-peroxide-producing NAD(P)H-oxidase, which are essential enzymes for the iodination of thyroglobulin, was cytochemically determined in the thyroid follicular cells of propylthiouracil (PTU)-treated rats. Peroxidase activity was determined using the diaminobenzidine technique. The presence of NAD(P)H-oxidase was determined using H2O2 generated by the enzyme; the reaction requires NAD(P)H as a substrate and cerous ions for the formation of an electron-dense precipitate. Peroxidase activity was found in the developed rough endoplasmic reticulum (rER) and Golgi apparatus, but it was also associated with the apical plasma membrane; NAD(P)H-oxidase activity was localized on the apical plasma membrane. The presence of both enzymes on the apical plasma membrane implies that the iodination of thyroglobulin occurs at the apical surface of the follicular cell in the TSH-stimulated state which follows PTU treatment.     

Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.     

Ultrastructural aspects of the goiter in cog/cog mice

Thyroids of congenitally goitrous (cog/cog) mice were studied with light and electron microscopy. The principal alteration in follicular cells was their largely overdistended rough endoplasmic reticulum (RER). Our findings resemble the ultrastructural features of human hypothyroid goiter caused by a thyroglobulin (TG) defect and thus support the previously suggested abnormalities of TG synthesis and/or processing in cog/cog mice.     

Differentiation and Ultrastructure of the Protophloem Sieve Elements of Minor Veins in the Maize Leaves: Changes in the Protoplast

Protophloem sieve element differentiation in the minor veins of the maize ( Zea mays L. ) leaves was first evidenced as an increase of the wall thickness, which began in the comers of the cell and then extended to other parts of the wall, and the appearance of long rough endoplasmic reticulum cistemae distributed throughout the cytoplasm, and then the presence of characteristic crystalloid inclusions within the plastids. As differentiation progressed, long cisternae of rough endoplasmic reticulum appeared to transform into shorter forms and eventually aggregated into small stacks, losing their ribosomes during the process. The nuclei degenerated, although frequently persisted until very late in differentiation the stages of maturation, as darkly stained amorphous aggregates surrounded by double nuclear envelope or only inner membrane of nuclear envelope. Subsequently, the nuclear envelope collapsed and became discontinuous. At the beginning of nuclear degeneration the perinuclear spaces were partly dilated and sometimes the outer nuclear envelope in the dilated portions then ruptured, and was accompanied by the disappearance of the cytoplasmic portion near it. During the peried of nuclear degeneration, in addition to the endoplasmic reticulum, plastids and mitochondria underwent structural modification, while components such as ribosomes, cytoplasmic ground substances, vacuoles and dictyosomes disintegrated and disappeared. At maturity, the surviving protoplasmic components, including plasmalemma, mitochondria, small stacked smooth endoplasmic reticulum and P-type plastids with crystalloids, became parietal in position. As differentiation of adjacent metaphloem sieve elements proceeded, the protoplasmic components of the mature protophloem sieve elements progresively degenerated and finally obliterated.     

Inhibition of thyroid-restricted genes by follicular thyroglobulin involves iodinated degree

Follicular thyroglobulin (TG) reflects the storage of both iodine and thyroid hormone. This is because it is a macromolecular precursor of thyroid hormone and organic iodinated compound in follicular lumen. Thus, it may have an important feedback role in thyroid function. In this study, monolayer cells were cultured and follicles were reconstituted with primary pig thyroid cells in vitro. Reconstituted follicles were treated with iodine and methimazole (MMI), a drug that blocks iodine organification and reduces the degree of TG iodination in follicular lumen. The high degree of iodinated TG in follicular lumen was observed to inhibit thyroid-restricted gene expression. To confirm this finding, monolayer thyroid cells were treated with a different degree of TG iodination at the same concentration. These iodinated TG were extracted from reconstituted follicles of different groups. In this manner, this study provides firsthand evidence suggesting that follicular TG inhibits the expressions of thyroid-restricted genes NIS, TPO, TG, and TSHr.     

Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).     

Elevated blood Hsp60, its structural similarities and cross-reactivity with thyroid molecules,and its presence on the plasma membrane of oncocytes point to the chaperonin as an immunopathogenic factor in Hashimoto's thyroiditis

The role Hsp60 might play in various inflammatory and autoimmune diseases is under investigation, but little information exists pertaining to Hashimoto’s thyroiditis (HT). With the aim to fill this gap, in the present work, we directed our attention to Hsp60 participation in HT pathogenesis. We found Hsp60 levels increased in the blood of HT patients compared to controls. The chaperonin was immunolocalized in thyroid tissue specimens from patients with HT, both in thyrocytes and oncocytes (Hurthle cells) with higher levels compared to controls (goiter). In oncocytes, we found Hsp60 not only in the cytoplasm but also on the plasma membrane, as shown by double immunofluorescence performed on fine needle aspiration cytology. By bioinformatics, we found regions in the Hsp60 molecule with remarkable structural similarity with the thyroglobulin (TG) and thyroid peroxidase (TPO) molecules, which supports the notion that autoantibodies against TG and TPO are likely to recognize Hsp60 on the plasma membrane of oncocytes. This was also supported by data obtained by ELISA, showing that anti-TG and anti-TPO antibodies cross-react with human recombinant Hsp60. Antibody-antigen (Hsp60) reaction on the cell surface could very well mediate thyroid cell damage and destruction, perpetuating inflammation. Experiments with recombinant Hsp60 did not show stimulation of cytokine production by peripheral blood mononuclear cells from HT patients. All together, these results led us to hypothesize that Hsp60 may be an active player in HT pathogenesis via an antibody-mediated immune mechanism.     

Myelin-like structures as a possible source of the smooth endoplasmic reticulum in early amphibian oocytes

A comparative study of amphibian oocyte ultrastructural organization has shown a significant accumulation of elements of the smooth endoplasmic reticulum in the oocyte cytoplasm at the third stage of development. The analysis of oocytes of two frog species, Xenopus laevis and Rana temporaria, at the first and second stages of their development enabled us to recognize in the cytoplasm of the oocyte some myelin-like structures (MLs) made of 30-40 densely packaged membranous layers and shaped as dense bodies. MLs are also present in the adjacent follicular cells and in the intercellular space. In the oocyte cytoplasm these structures are located near the nuclear envelope and other intracellular organelles. At the third stage of oogenesis, which is characterized by a high functional activity of the cells, MLs are seen to unwrap sequentially into double-layer membranes similar to the smooth endoplasmic reticulum cisternae. Intermediate steps of this process being also observed. It is supposed that MLs may play the role of membrane stocks to be used eventually for the formation of nascent endoplasmic membranes in the amphibian oocytes.     

The nuclear reticulum in placental cells ofLilium regale is a part of the endomembrane system

Summary Placental cells line the ovarian transmitting tract inLilium regale and produce exudates for secretion. Sections through the highly lobed nuclei of these cells reveal the presence of membrane profiles which form vesicles with varying dimensions in cross section. Computer reconstruction of the nucleus reveals that the vesicle profiles form a complex reticulum of tubular cisternae, which spans the whole nucleus, enclosing a maze of continuous lumen space. Connections between the vesicles and the inner nuclear envelope are visible at various points along the nuclear envelope. This complex network of tubules which constitutes the reticulum arises from the inner nuclear membrane. The nuclear reticulum dramatically increases the inner-envelope surface area, comprising 82% of the total membrane perimeter of inner nuclear envelope and nuclear reticulum. The inner nuclear envelope invaginates into the nucleus forming the nuclear reticulum and the outer nuclear envelope evaginates into the endoplasmic reticulum (ER), indicating that there is a continuity between the lumens of the nuclear reticulum and the ER. The nuclear reticulum is labelled with zinc iodide-osmium tetroxide, a staining pattern identical to that seen in the ER. Positive reaction to the zinc iodide-osmium tetroxide indicates that the nuclear reticulum is a site for Ca²⁺ deposition. The nuclear reticulum forms an extension of the endomembrane system which reaches deep into the nucleoplasm. The lumenal continuity of this system means that there is a channel for communication from the cytoplasm into the nucleoplasm, and that this channel sequesters calcium.Abbreviations ER endoplasmic reticulum - TEM transmission electron microscope - ZIO zinc iodide-osmium tetroxide     

Development of a nuclear polyhedrosis virus in midgut cells and penetration of the virus into the hemocoel of the armyworm,Pseudaletia unipuncta

The development of a nuclear polyhedrosis virus (NPV) in larval midgut cells of the armyworm, Pseudaletia unipuncta, is similar to that of other NPV. In the nucleus, the envelopes around the nucleocapsids seem to be derived de novo or from the inner layer of the nuclear envelope wich forms cisternae, blebs, or infoldings. The nucleocapsids are also enveloped by synhymenosis during passage through the nuclear membrane, the cell membrane, or the endoplasmic reticulum membrane. Both enveloped and unenveloped nucleocapsids may enter the cytoplasm through the nuclear pore or budding through the nuclear membrane. From the cytoplasm the virions may enter the hemocoel through the basal cell and basement membranes or through the endoplasmic reticulum, intercellular space, and the basement membrane.     

Endogenous peroxidase activity in mononuclear phagocytes

The diaminobenzidine (DAB) technique has been used to visualize the subcellular localization of peroxidatic enzymes in mononuclear phagocytes. The latter cells are part of the mononuclear phagocyte system (MPS), which includes the monocytes in the bone marrow and blood, their precursors in the bone marrow, and the resident macrophages in the tissues. The DAB cytochemistry has revealed distinct subcellular distribution patterns of peroxidase in the mononuclear phagocytes. Thus the technique facilitates the identification of the various phagocyte types: Promonocytes contain peroxidase reaction in the nuclear envelope, endoplasmic reticulum, Golgi apparatus, and cytoplasmic granules. Monocytes exhibit the reaction product only in cytoplasmic granules. Most resident macrophages show the activity only in the nuclear envelope and endoplasmic reticulum. Furthermore, new phagocyte types have been detected based on the peroxidase cytochemistry. Intermediate cells between monocytes and resident macrophages contain reaction product in the nuclear envelope, endoplasmic reticulum and cytoplasmic granules. The resident macrophages can be divided into two subtypes. Most of them exhibit the pattern noted above. Some, however, are totally devoid of peroxidase reaction. Most studies on peroxidase cytochemistry of monocytes and macrophages agree that the peroxidase patterns reflect differentiation or maturation stages of one cell line. Some authors, however, still interpret the patterns as invariable characteristics of separate cell lines. As to the function of the peroxidase in phagocytes, the cytochemical findings imply that two different peroxidatic enzymes exist in the latter cells: one peroxidase is synthesized in the endoplasmic reticulum of promonocytes and transported to granules via the Golgi apparatus. The synthesis ceases when the promonocyte matures to the monocyte. Upon phagocytosis the peroxidase is discharged into the phagosomes. Biochemical and functional studies have indicated that this peroxidase (myeloperoxidase) is part of a microbicidal system operating in host defence mechanisms. The other enzyme with peroxidatic activity is confined to the nuclear envelope and endoplasmic reticulum of resident macrophages in-situ and of monocytes at early stages in culture. As suggested by the subcellular distribution, the inhibition by peroxidase blockers, and the localization during phagocytosis studies, the latter peroxidase is functionally different from the myeloperoxidase.(ABSTRACT TRUNCATED AT 400 WORDS)