Fig 1.
Light microscopy of the pituitary in situ and histological sections of the gland.
(a) A ventral view of the brain showing position of the pituitary and the different nervous tissues that surround it. (b) Dorsal view of the pituitary and the hypothalamus showing the third brain ventricle. (c) A sagittal view of the pituitary and the adjoining tissues. (d) A middle sagittal section of the pituitary at the most rostral side showing its connection with the hypothalamus. (e) The same pituitary section as (d) but showing the dorsal border where a membrane is observed covering the gland. (f) A detail of the dorsal surface of the PN and the external membrane. (g) A detail of the dorsal surface of the PI showing a space between two lobules and the membrane at the external side. (h) A detail of the border of the PD and the external membrane. Arrow, blood vessel; thin empty arrow, pituitary stalk; empty arrowhead, optic chiasm; arrowhead, dura mater; thick empty arrow, fibrous membrane; asterisk, pituitary cleft. PI, pars intermedia; PN, pars nervosa; PD, pars distalis; PT, pars tuberalis; H, hypothalamus; B, brain; MB, midbrain; SB, sphenoid bone; C, capsule; Fr, frontal; Ca, caudal.
Fig 2.
Scanning electron microscopy (SEM) of the exterior of the pituitary gland.
(a) A view at low magnification of the posterior face of the pituitary where an external envelope or capsule looks like a thickness layer covering the PI, a furrow delimits the PN and PI borders, and the thicker external envelope becomes thinner at PD. (b) An approach of the zone marked by the square in (a) to illustrate the previous description. The dashed line square is included and described in Fig 2(A). (c) High magnification of the capsule that folds and looks if it surges or invaginates from the border of the PI in the vicinity of PN. (d) A detail of the zone where the PI and PN are close together, showing the furrow between them and the covering or invagination of the thicker capsule of both sides. (e) A view of the PD surface. (f) High magnification of the square in (e), where some erythrocytes are seen on the surface, but more relevant is the presence of abundant blood vessels. Arrow, blood vessels; PI, pars intermedia; PN, pars nervosa; PD, pars distalis; C, capsule.
Fig 3.
Characteristics of the capsule that covers pars intermedia.
(a) SEM of the capsule detail (dashed line square from Fig 2(B) that covers the PI. (b) High magnification of the rectangle in (a) to appreciate the folded and fibrous aspect of the pituitary capsule. Note the digitiform protrusions and the multiple fibrous layers. (b) Higher magnification of the surface of a capsule fold with digitiform protrusions and showing multiple fibrous layers. (c) Semithin sections of the PI covered by the capsule, which is similarly to lose connective tissue. The PI consists of a relatively large amount of amorphous extracellular matrix with capillaries and different types of cells. Moreover, the PI and the capsule are separated by a thin fibrous layer and fibroblast-like cells from the capsule, whose external limit looks denser and thicker, with fusiform cells and peripheral capillaries. Some erythrocytes were observed on the capsule surface. The rectangle is observed as a higher magnification of the capsule in (d). (e) Transmission electron microscopy (TEM) of the loose connective tissue with fibrocytes forming well-defined and clear spaces where macrophages-like cells with cytoplasmic protrusions were frequently observed. Other less differentiated cells with large nuclei and abundant euchromatin were located outside the spaces. (f) TEM of the external limit of the capsule showing lamellar organization of overlapping cytoplasmic prolongations from fibrocytes. Long and flattened fibroblast-like cells were observed at the outermost layer of the capsule. These connective tissue elements surrounded the large diameter and sinuous capillaries. (g) Some places of the outermost lamellae of the capsule had fusiform fibroblast-like cells limiting clear and well-defined spaces. (h and i) High magnifications of overlapping cytoplasmic prolongations from fibrocytes, showing numerous mitochondria and interdigitations. Arrow, capillary; arrowhead, fibroblast-like cell; thick arrow, fibrous layers; PI, pars intermedia; C, capsule.
Fig 4.
Characteristics of the pituitary capsule covering the dorsal-lateral side of the pituitary gland.
(a) SEM of the multiple-layered structure of the capsule is evident, bordering the anterior epithelium of the pituitary cleft and covering the PD. (b) A higher view of the rectangle in (a). (c) SEM of the capsule at the lateral side of the PD showing its multiple-layered, surface cells and a capillary. (d) SEM of the lateral aspect of the PD showing the site where the capsule was torn. (e) A higher magnification of the square in (d) shows the exposure, with numerous cells protruding towards the surface, (f) superficial PD border showing cells with smooth surface some of which show very short primary cilia-like structure, as it is observed at higher magnification in (g and h). (i and j) Semithin sections of the outer surface of the PD showing the multilayered capsule, with embedded capillaries and fibroblast like cells, which is thicker and more complex towards the dorsal lateral aspect of the PD. (k) A view more at the dorsal-lateral aspect than in (j) but also displays organization with invaginations at its more dorsal surface. Note the clear multilayered fine aspect and the space formation in the capsule; these spaces contain lymphocyte- and macrophage-like cells (l and m). Arrow, capillary; thick arrow, fibrous layers; empty arrow, cilium; arrowhead, fibroblast-like cell; asterisk, anterior face of the cleft; PD, pars distalis.
Fig 5.
Characteristics of the PD and the pituitary capsule at the ventral side of the gland.
(a) SEM of the surface of a PD lateral wing at its more ventral side showing a sinuous and folded appearance. (b) At higher magnification, it is possible to observe the fibrous and reticular material that constitutes the capsule. (c) A semithin section showing the cellular cord arrangement and different sizes of capillaries of the PD, and at the external surface, a thin mesh of fibroblast-like cells and reticular fibres with capillaries embedded in it. (d) TEM of the lateral border of the ventral side of the PD showing an envelope or capsule form by an extracellular matrix with fibrillar components and by a thin continuous amorphous sheet that closely follows the basal contour of endocrine cells. (e) A higher magnification of the square in (d) showing that this sheet is formed by a lamina densa separated from the cell surface by a lamina lucida or lamina rara. (f) A detail of long and slender, parallel, cytoplasmic processes from fibroblast-like cells of the capsule. The lamina basal appears to be discontinuous. (g) The capsule, including the lamina basal, was also observed covering two agranular cells. Arrow, capillary; arrowhead, fibroblast-like cell; empty arrowhead, lamina basal; empty arrow, cytoplasmic process; G, secretory cell; FS, agranular cell.
Fig 6.
Characteristics of the PI-side epithelial lining of Rathke´s cleft.
(a) SEM of the PI and PN and the epithelia of the pituitary cleft as a cuboid layer. (b) Higher magnification of the epithelial layer showing that the main cell type of this epithelium is the multiciliate cell. (c) This epithelium changes at the medial zone, where fewer multiciliate cells are observed. There are cells with microvilli in dome-like apical domain and a third cell type with microfolds and a primary cilium was identified (d). (e and f) Semi thin sections showing the organization of the epithelia as columnar pseudostratified with multiciliate cells and cuboidal cells. The underlying tissue is a scarce lamina propria or stroma with capillaries, pericytes and fibroblast-like cells, that separates the epithelium and endocrine cells. Connective tissue septa from the stroma are frequent, dividing the endocrine tissue into lobules. (g) TEM of the posterior epithelia lining the cleft shows columnar pseudostratified organization, where basal mitochondria-rich cells are covered by apical electron-dense multiciliate cells and cells with microvilli. (h) Details of the lateral cell membranes of the apical epithelial cells showing the junctional complexes; no adhesion complexes were observed between the epithelial cells and the cells below them. Arrow, capillary; thick arrow, cilia; empty arrow, cilium; empty arrowhead, microvilli; arrowhead, fibroblast-like cell; empty thick arrow, cell-cell adhesion complex; m, mitochondria; thin arrowhead, pericyte; PI, pars intermedia; PN, pars nervosa.
Fig 7.
Characteristics of the PD-side epithelial lining of Rathke´s cleft.
(a and b) SEM show a surface decorated with apexes of different sizes, some with microvilli irregular in size and shape, other with tufts of cilia, and others rounded and smoother. Flattened areas were also noted at this surface of the Rathke´s cleft. the border of the PD wing and the epithelial cleft layer. (c) Some apexes presented a primary cilium-like structure. (d) A semithin section of the epithelia showing two types of columnar cells, ciliated and with microvilli, whose nuclei are arranged in rows parallel to each other, and some small, superficial cuboidal to flattened cells, with rounded nuclei, are identified. (e) TEM of the epithelial cells where a multiciliate cell, recognized by its basal body, is between two cells with microvilli plentiful mitochondria. The upper right panel shows details of the cell-cell adhesion complexes among epithelial cells. (f) TEM shows cells with irregular shapes and sizes of microvilli, with abundant ribosomes, and some exhibit large droplets of colloidal material. The cell-cell adhesion complexes present in these cells are composed of tight junctions, adherence junctions and desmosomes. Arrow, capillary; thick arrow, cilia; empty arrow, cilium; empty arrowhead, microvilli; arrowhead, colloidal droplet; empty thick arrow, cell-cell adhesion complex; asterisk, anterior face of the cleft; m, mitochondria; PD, pars distalis.
Fig 8.
Localization of red fluorescent Evans blue in the pituitary after intracardiac or intracerebroventricular administration of the dye, and of NaFluo administered via intracerebroventricular.
(a) Presence of Evans blue at the periphery of the PN and (b) at the periphery of the PD, after the cardiac administration of Evans blue, and the rat nose and ears were bluish. (c and d) Ten minutes later after the intracerebroventricular administration of Evans blue, the dye is observed at the PN meninges, the pituitary cleft, and the PD periphery. (e and f) Two views of the PD surrounded by Evans blue dye intracerebroventricularly administered. (g and h) Two views of the accumulation of NaFluo 40 minutes later after it´s intracerebroventricular administration and followed by Evans blue administered via intracardiac. Evans blue stains in red (excitation = 561 nm, emission = 576/651 nm) and NaFluo in green (excitation = 488 nm, emission = 510/548 nm). Z-projection: (a) 130 μm, (b) 80 μm, (c) 100 μm, (d) 120 μm, (e) 120 μm, (f) 120 μm, (g) 70 μm, (h) 50 μm. PN, pars nervosa; PI, pars intermedia; PD, pars distalis; asterisk, pituitary cleft.
Fig 9.
Presence of fluorescent beads and NaFluo accumulation in the pituitary gland after their intracerebroventricular administration.
(A) Localization of 2- and 0.2-μm diameter fluorescent beads in the pituitary gland after intracerebroventricular administration. (a) A confocal view of the pituitary where 2-μm diameter fluorescent beads are observed at the PN periphery and inside the pituitary cleft. (b) A confocal view of the PD periphery showing 2-μm diameter fluorescent beads. (c) A confocal view of the periphery of the PD showing an accumulation of 2- and 0.2-μm diameter fluorescent beads. The polystyrene beads are green (excitation = 488 nm, emission = 510/548 nm), the cell nucleus in blue (excitation = 405 nm, emission = 413/479 nm). Z-projection: (a) 94 μm, (b) 36 μm, (c) 27 μm.(d) A view of a pituitary sagittal section stained with HE shows cells inside Rathke´s cleft. (e) Higher magnification of the cells inside Rathke´s cleft. (f) Fluorescens microscopy of a cell at the pituitary cleft that phagocytosed 2- and 0.2-μm diameter fluorescent beads, 20 μm Z-projection. PN, pars nervosa; PI, pars intermedia; PD, pars distalis; asterisk, pituitary cleft. (B) Accumulation of NaFluo after 10- and 20-min of intracerebroventricular administration in different intracranial tissues: Pituitary; ME, media eminence; CE, cerebellar cortex; ON, optic nerve. Differences in fluorescence between the tissue were analyzed by one-way analysis of variance (ANOVA) followed by Holm-Sidak´s multiple comparisons test. Differences between endogenous and NaFluo accumulation MR and AR groups, * P <0.05, ** P <0.01, ** P <0.0001.