Effect of drugs on pituitary ultrastructure.

Various drugs and hormones influence the light microscopic and especially the electron microscopic structure of the anterior pituitary and its tumors. Many structural effects are known only from animal experiments since specimens from human pituitaries are mostly not available. The structure of growth hormone (GH) cells is relatively stable. A massive GH cell hyperplasia is known only in rare cases with growth hormone releasing factor (GRF) excess from tumors. Prolactin cells can be stimulated by drugs, neurotransmitters, and hormones which decrease the dopamine inhibition. Adrenocorticotropic hormone (ACTH) cells are stimulated by stress, some hormones, loss of adrenals, and drugs which activate the alpha 1- and beta-receptors or inhibit the alpha 2-receptors. They are suppressed and changed into Crooke's cells by treatment with glucocorticoids. Thyroid-stimulating hormone (TSH) cells increase in number and size in states for overstimulation especially by thyrotropin releasing hormone (TRH). A decrease results from hyperthyroidism and possibly from somatostatin, L-dopa, and dopamine. Gonadotroph cells transform into castration cells in strongly hyperactive states (gonadectomy, antiandrogens, gonadotropin releasing hormone [Gn-RH]agonists, aminoglutethimide). Special types of pituitary adenomas can be treated with drugs which suppress hormone production and proliferation. Dopamine agonists and somatostatin reduce the tumor size of varying proportions of GH secreting adenomas in acromegaly. Ultrastructurally, a decrease of cytoplasmic and nuclear volume and an increase of lysosomes are found. Bromocriptine and other dopamine agonists are established in the treatment of prolactin secreting adenomas. They induce a shrinkage in many cases. Ultrastructurally, a reduction of cellular and nuclear size, an increase in number of secretory granules and of lysosomes, and a reduction of rough endoplasmic reticulum can be demonstrated.     

Angiogenesis in pituitary adenomas

Angiogenesis, which is the formation of new blood vessels from preexisting capillaries, plays an important role in tumor growth and metastasis. In this review, the focus is on angiogenesis in pituitary adenomas. Angiogenesis of pituitary adenomas has been assessed by studying tumor microvessel density using specific immunohistochemical markers to clarify the relationship between angiogenesis and tumor behavior. Unlike other organs, pituitary adenomas have significantly lower vascular densities as compared to nontumorous adenohypophysis, suggesting that the lack of significant angiogenesis may play a role in the slow pace of pituitary tumor growth and rarity of metastases. In addition, the relationship between microvessel density and various factors in pituitary adenomas is reviewed, including tumor types, age and sex, invasiveness, malignancy, several proliferative markers (MIB-1 or Ki-67). However, further studies will be needed, since many studies have reached opposite conclusions. Angiogenesis is a complex multistep process and several factors are found to be involved in each step of neovascularization, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and various other cytokines. VEGF and bFGF (or FGF-2), which are the most potent angiogenesis inducers among them, have been studied by immunohistochemistry, in situ hybridization, or in vitro studies in normal or adenomatous pituitaries and these results are also discussed in this review.     

Use of ultrastructural immunohistochemistry in human pituitary pathology.

Recent advances in ultrastructural immunohistochemistry have provided insight into not only the subcellular localization of single antigens but also the colocalization of two distinct antigens in the same cellular constituent. In the field of pituitary pathology, precise identification of cell types, mechanism of processing, and dynamic intracellular transportation of hormones, as well as production of multiple hormones in the same cells of nontumorous and neoplastic adenohypophyses, have been documented by use of these techniques. The present review deals with the use of major methods for ultrastructural immunohistochemistry including pre-, post-, and non-embedding methods, particularly focusing on their application to human pituitary pathology. Problems of tissue processing and a protocol for double labeling technique using the protein A-gold complex are also described.     

Ultrastructural immunocytochemistry of the adenohypophysis in the rat: A review

Immunocytochemistry has made great strides in the morphology of endocrine glands, especially the adenohypophysis, because the localization of hormones can be clearly demonstrated by this method in the microscopic preparations both for light and electron microscopy. In the adenohypophysis, electron microscopic immunocytochemistry is useful for identifying the producer cell of each hormone. The second contribution is its application to the cell biology of secretion mechanisms. The pituitary hormones, their precursors, derivatives, and fragments were artificially synthesized and their antibodies were produced. Using these antibodies the intracellular sites of synthesis, condensation, processing, and sorting were studied under the electron microscope. The ultrastructure of each cell organelle and its alteration due to the changing function was studied. It was proved that the intracisternal granules in the thyroidectomy cells contain thyroid-stimulating hormone (TSH). The trans-Golgi network or GERL contains a peculiar supporting structure, intracisternal skeleton. Transport of secretory granules may be performed in relation to the microtubules, actin, and some related substances. The most frequently observed mode of hormone release in the adenohypophysis is exocytosis. Sometimes multigranular exocytosis occurs. Vesiculation of membrane around the secretory granules often occur inward or outward. The inward vesiculation forms pinocytotic vesicles, through which the membrane material may be retrieved. The outward vesiculation forms vesicle-like fragments of cytoplasm being discarded to the extracellular space. By these mechanisms the surface area of the cell is maintained constantly.     

Ultrastructural diagnosis of human pituitary adenomas.

Electron microscopy, which has been instrumental in the characterization of normal pituitary cell types, has also played a crucial role in the morphologic classification of pituitary adenomas arising in the presently known 5 cell types, and in the recognition of 3 adenoma types with yet undisclosed cell derivation. This review deals with the application of electron microscopy for study of pituitary adenomas in order to provide specific pathological diagnosis and aid the clinician in selecting appropriate postoperative treatment. In addition to the ultrastructural appearance and diagnostic features of 15 adenoma types, the morphology of hyperplastic proliferations and that of known normal counterparts of various adenoma types are also discussed. Specific morphologic diagnosis of pituitary lesions is important not only for adequate postoperative management of patient, but is also a prerequisite for study of the natural history and biological behaviour of various adenoma types.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.     

Cell biology of the adipokinetic hormone-producing neurosecretory cells in the locust corpus cardiacum.

The adipokinetic cells are neuron-like unipolar cells, the cell bodies and cell processes of which are intermingled within the glandular part of the corpus cardiacum. In Schistocerca gregaria, they produce two adipokinetic hormones, AKH-I and -II, whereas in Locusta migratoria an additional hormone, AKH-III, is present. The three AKHs are produced by the same cells and are co-localized in secretory granules. The biosynthesis and processing of the AKH prohormones to the bioactive hormones, which has been elucidated in detail for AKH-I and -II in S. gregaria, takes less than 75 min and goes on continuously. In older locusts in particular, the adipokinetic cells contain intracisternal granules, widely dilated cisternae of the rough endoplasmic reticulum, which function as stores of prohormones of AKH-I and -II, not of AKH-III. The adipokinetic cells are subjected to regulation by a number of neural and humoral substances, neural influences coming from secretomotor cells in the lateral part of the protocerebrum. Flight activity is the only natural stimulus unequivocally shown to induce the release of AKHs, which in L. migratoria results in parallel secretion of all three AKHs. During secretory stimulation, young secretory granules containing newly synthesized hormones are preferentially released over older granules. Secretory stimulation is not accompanied by a clear increase in the levels of the AKH mRNAs and the AKH prohormones and in the rate of synthesis of the (pro-)AKHs. Apparently, a coupling between release and biosynthesis of the AKHs in the adipokinetic cells is very loose or does not even exist.     

Vascular networks and endothelial cells in the rat experimental pituitary glands and in the human pituitary adenomas

There has been considerable interest in the relationship between hormone-secreting endocrine cells (HSEC) and their microvessels (MVN) in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). Therefore, this study was designed to visualize and to reveal the relationship between hormone secreting endocrine cells and their microvessel environment including vascular endothelial cells in 3D using rat pituitary glands under various experimental conditions by confocal laser scanning microscopy (CLSM). By CLSM, the 3D distributions of MVN were visualized and revealed a relationship between HSEC and MVN in experimental pituitary glands and human pituitary adenomas. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.     

Morphological identification of pituitary hormones by the aid of pH-determination using microphotometry (author's transl)

The identification of pituitary ACTH and TSH producing cells was performed by histological staining at the isoelectric points (pH levels: 4.6 and 5.2 respectively) of these hormones in cat. Intensity of stained material in the hormone producing cells was determined by microphotometric technique. By the application of hormones influencing the pituitary ACTH and TSH producing relevant changes in pituitary cells could be detected.     

Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures

Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described.     

Sensilla and secretory glands in the antennae of a primitive ant: Dinoponera lucida (Formicidae: Ponerinae)

Morphology of the antennae of the female workers of the ponerine ant Dinoponera lucida was examined by light and scanning electron microscopy. In several antennomers, we found secretory gland cells of class I and III. Class III gland cells release their secretion through single pores in the antennal surface, whereas class I secretory cells are seen as tall epidermal cells close to the cuticle. Both gland types have weak reaction for total proteins and neutral polysaccharides. Six distinct sensilla types were observed: trichodea, chaetica, campaniform, basiconica, placodea, and coeloconica. The possible sensory functions of these sensilla and the gland functions are discussed.     

Review: Studies on the reproductive and developmental biology of <I>Cichlasoma dimerus</I> (Percifomes,Cichlidae)

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.     

Hindbrain floor plate of the rat: ultrastructural changes occurring during development

Most of the molecular and experimental studies on the floor plate (FP) have been performed on the FP region extending along the spinal cord. However, little is known about the hindbrain FP. The FP undergoes regional and temporal changes throughout development, but information with respect to the ultrastructural correlate of such changes is missing. The present investigation was focused on the ultrastructural developmental changes occurring in the FP of the rat hindbrain. The FP cells of the hindbrain secrete a material reacting with antibodies against the secretory glycoproteins of the subcommissural organ (AFRU). This antibody was used to perform an ultrastructural immunocytochemical analysis of the rat FP. From E-12 on, there is a progressive increase in the development of the rough endoplasmic reticulum (RER), so that by E-18, it has reached a high degree of hypertrophy. A unique feature of the hindbrain FP cells is the presence of tubular formations and 140-nm vesicles that appear to originate from RER cisternae. The labelling of these two structures with AFRU and Concanavalin A strongly suggests that they are pre-Golgi compartments containing secretory material. Since these structures are present in the basal process and in the apical cell pole of the FP cells, the possibility that they release their content at these sites, is discussed. It is proposed that a secretory mechanism bypassing the Golgi apparatus (constitutive secretion?) operates in the FP cells. The presence of apoptotic cells within the FP of E-20 embryos and newborns suggests that death, and not re-differentiation, is the fate of the FP cells.     

Proteomics of regulated secretory organelles

Regulated secretory organelles are important subcellular structures of living cells that allow the release in the extracellular space of crucial compounds, such as hormones and neurotransmitters. Therefore, the regulation of biogenesis, trafficking, and exocytosis of regulated secretory organelles has been intensively studied during the last 30 years. However, due to the large number of different regulated secretory organelles, only a few of them have been specifically characterized. New insights into regulated secretory organelles open crucial perspectives for a better comprehension of the mechanisms that govern cell secretion. The combination of subcellular fractionation, protein separation, and mass spectrometry is also possible to study regulated secretory organelles at the proteome level. In this review, we present different strategies used to isolate regulated secretory organelles, separate their protein content, and identify the proteins by mass spectrometry. The biological significance of regulated secretory organelles‐proteomic analysis is discussed as well. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:844–867, 2009     

Physiological and pathological angiogenesis in the endocrine system

Formation of new blood vessels occurs in many physiological states (during development of the embryo, cycling changes of the female reproductive tract), as well as in pathological processes (such as diabetic retinopathy and wound healing). Angiogenesis has been shown to be related to tumor formation, prognosis, and response to treatment in many tumor types. Intratumoral microvessels can be related to tumor behavior or hormone secretion in different endocrine tumors. For example, invasive prolactinomas are more vascular than noninvasive adenomas; a surgical approach is more successful in macroprolactinomas with lower microvessel density. A higher number of microvessels have been found in papillary thyroid carcinomas during recurrences. A correlation between microvessel count and prognosis in papillary and medullary thyroid carcinomas has been suggested. Several stimulating and inhibiting factors involved in the regulation of angiogenesis have been identified. Among them, vascular endothelial growth factor (VEGF) has been shown to be critically involved in angiogenesis and also in the neovascularization of solid tumors. Dopamine agonists (already in clinical use for prolactinomas) have potent inhibitory actions on VEGF signaling, and thus may be a new tool in antiangiogenic therapy. Secretion of VEGF in the great majority of human pituitary adenomas is inhibited by dexamethasone. This suggests that glucocorticoids can be considered in the treatment of certain pituitary tumors. The cyclic nature of angiogenesis in the female reproductive tract indicates that stimulation or inhibition of paracrine angiogenic factors may lead to new approaches for being able to influence reproductive endocrine disorders. Experimental and clinical aspects of interactions between angiogenic factors and tumor growth of the endocrine system are also discussed.     

Effects of drugs on pituitary fine structure in laboratory animals

Although an increasing number of chemicals are reported to affect endocrine glands, only a few studies are dealing with their toxic effect on pituitary. The drugs can induce lesions acting directly on endocrine cells or indirectly by interfering with the regulation of their endocrine activities. Some drugs stimulate pituitary cell proliferation leading to hyperplasia and tumor formation; other chemicals have an inhibitory effect on adenohypophysial cells; and only one drug, hexadimethrine bromide, has been found to induce pituitary necrosis. Although complex toxicologic studies have been carried out on many chemicals, the mechanism of action of most drugs is not completely elucidated and further studies are necessary to establish structure function correlations.     

Fine structural cytology of the adenohypophysis in rat and man

The present review deals with the use of electron microscopy in the identification of pituitary cell types as well as the assessment of their functional state, in rat and man. Application of immunoelectron microscopy, especially immunogold techniques, utilizing multiple labeling in establishing differentiation and hormone content of cell types, is emphasized. Recent evidence of plurihormonality in various pituitary cell types indicates that the once axiomatic one cell-one hormone theory is untenable and that the present perception of pituitary cell types and their function requires modification. Detection of hormonal and nonhormonal substances in pituitary cell types, not associated with their known endocrine function, suggests that hypophysial cells may have yet unknown roles, possibly in the realm of paracrine and autocrine regulation.     

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.