Folliculostellate cells in pituitary pars distalis of male viscacha: immunohistochemical, morphometric and ultrastructural study

Abstract

Folliculostellate cells (FSC) have been reported in pituitary of several mammalian species. FSC morphology and secreted substances have been instrumental to the understanding of their function. The purpose of this work was to perform an immunohistochemical, morphometric and ultrastructural study of the pituitary pars distalis FSC in adult male viscacha and to analyze their relation with hormone secreting cells. Immunohistoche-mistry and image analysis were carried out in different sectors of the gland, from the middle (sector 1) to the glandular periphery (sector 5). Transmission electron microscopy with lanthanum as electrodense tracer was used. FSC formed follicles with PAS-positive colloid inside. They expressed S-100 protein mainly in both nucleus and cytoplasm. FSC were stellate-like in shape and exhibited short cytoplasmic processes that contacted with blood vessels and endocrine cells. In addition, some follicular colloids were immunostained with anti-S-100 protein. A few FSC were immunostained with anti-glial fibrillary acidic protein (GFAP) and anti-vimentin. The morphometric parameters analyzed (percentages of S-100-positive total, cellular and colloidal areas) increased from sector 1 to sector 3 and then decreased to sector 5. Hormone secreting cells, mainly lactotrophs, gonadotrophs and corticotrophs were associated with FSC and follicles. The ultrastructural study demonstrated that FSC developed junctional complexes and desmosomes between their lateral membranes. Lanthanum freely penetrated the spaces between granulated cells and FSC, but did not penetrate into the follicular lumen.

In conclusion: 1) the differential expression of S-100 protein, GFAP and vimentin may indicate different physiological stages of FSC; 2) the expression of these proteins suggests a neuroectodermic origin of these cells; 3) FSC spatial distribution, association with endocrine cells, and the generation of an intercellular communication network suggest that FSC are involved in the pituitary pars distalis paracrine regulation of the viscacha.

Figure 1

Recently removed pituitary of adult male viscacha captured in February (summer). Line M represents vertical cut to obtain two hemipituitaries, and the lines P and P' correspond to the glandular periphery extremes. The regularly spaced serial sections were grouped in five sectors (1 to 5) in each hemipituitary. Scale bar = 0.1 mm.

Figure 2

Sections of the pituitary of adult male viscacha stained with Hematoxylin-PAS technique. (A) The image shows: pars distalis (PD), pars intermedia (PI), pars nervosa (PN) and Rathke's pouch (r). The regions and extremes of the pituitary PD are shown: vr, ventral region; dr, dorsal region; ce, cephalic extreme; ca, caudal extreme. (B) Higher magnification of the upper insert in Figure A. Long blood vessels (v) branch out in PD are observed. Arrowheads: PAS-positive colloid. (C) The follicles with PAS-positive colloids inside (arrowheads) are surrounded by one or two layers of endocrine cells (arrows) and blood vessels (v). (D) Higher magnification of the lower insert in Figure A. Blood vessels (v) that communicate pars distalis (PD) with both pars intermedia (PI) and pars nervosa (PN) are observed. r: Rathke’s pouch. Scale bars: A = 500 µm; B and D = 100 µm; C = 25 µm.

Figure 3

Immunohistochemistry for S-100 protein (A–D), GFAP (E–F) and vimentin (G–H) in pituitary pars distalis of viscacha. (A) The immunostaining for S-100 protein is observed in the FSC and some follicular colloid (arrowheads) of pars distalis (PD) and pars intermedia (PI). The pituicytes (arrows) of pars nervosa (PN) also exhibit immunostaining for this protein. r: Rathke's pouch. (B) FSC (arrows) immunostained with anti-S-100 protein forming follicles surrounded by blood vessels (v). The follicular colloid (F) presents heterogeneous immunostaining for this protein. (C) Short cytoplasmic processes (P) of the FSC are in contact with blood vessels (v) and surround endocrine cells (arrowheads). F: immunostained follicular colloid. (D) Immunostaining for S-100 protein is observed in the nucleus (n), cytoplasm (c) and cytoplasmic process (P) of the FSC. The follicular colloid (F) exhibits a heterogeneous immunostaining pattern. v: blood vessel. Inset: A FSC (arrow) with only immunostained cytoplasm and other with a cytoplasmic process (P) surrounding an endocrine cell (arrowhead). (E–F) Scarce FSC (arrows) are immunostained with anti-GFAP in the cytoplasm around the nucleus. F: follicles without immunostaining for GFAP. Inset and Figure F, Short cytoplasmic processes (P) of FSC in contact with blood vessel (v) are observed. n: nucleus without immunostaining for GFAP. (G–H) A few FSC (arrows) show cytoplasmic immunostaining for vimentin, mainly in the cytoplasmic processes (P). The follicles (F) and nucleus (n) of FSC are not immunostained with anti-vimentin. (I) Negative control of immunoperoxidase staining. PD: pars distalis. r: Rathke's pouch. PI: pars intermedia. Scale bars: A = 250 µm; B-C = 12.5 µm; D-H and Insets = 5 µm; I = 100 µm.

Figure 4

Distribution of S-100 protein in pituitary pars distalis of adult male viscacha. Graph: The values are expressed as mean ± SEM. %IA S-100: percentage of S-100-positive total area; %IA S-100 cel: percentage of S-100-positive cellular area; %IA S-100 col: percentage of S-100-positive colloidal area. Significant differences were determined by analysis of variance followed by Tukey-Kramer multiple comparison test. %IA S-100: a, P<0.01; 3 versus 1 and 5. %IA S-100 cel: b, P<0.01; 3 versus 1 and 5. %IA S-100 col: c, P<0.01; 3 versus 5. The images show details of the pituitary cephalic extremes in the sectors 1 (A), 3 (B) and 5 (C) immunostained with anti-S-100 protein. In sector 3 (B) an increase of the immunopositive area is observed. Arrows: FSC. F: follicular colloids. Scale bars: A-C = 12.5 µm.

Figure 5

Micrographs of double-immunohistochemistry of anterior pituitary hormones (Prl, FSH, LH, ACTH, GH and TSH; brown) and S-100 protein (fuchsia). (A) A close association between lactotrophs (arrowheads) and FSC (arrow) originating follicles (F) are shown. Inset: Single-immunostaining for prolactin. Follicular colloid immunostained with anti-Prl (asterisk) and lactotrophs (arrowhead) near the follicle are observed. (B - Inset) The FSH-gonadotrophs (arrowheads) are contacting FSC (arrow) and follicles (F). v: blood vessel; n: S-100-positive nucleus of FSC. (C) Several LH-gonadotrophs (arrowheads) are associated to FSC (arrows) and follicles (F). Inset: The cytoplasmic process (P) of FSC is in contact with blood vessel (v). Asterisk: Follicular colloid immunostained with anti-LH. (D) The corticotrophs (arrowheads) are located near the FSC (arrows) and follicles (F). Inset: a cytoplasmic process (P) of FSC (arrow) surrounding a corticotroph (arrowhead) is observed. (E) Somatotrophs (arrowheads) exhibit a wide distribution in pituitary pars distalis, but they are only observed in the proximity of follicles (F). Arrow: isolated FSC with a short cytoplasmic process. Inset: Somatotroph (arrowhead), in the second layer limiting the follicle, and a cytoplasmic process (P) of FSC (arrow) reaching the blood vessel (v) are observed. (F - Inset). In the pars distalis caudal extreme, scarce thyrotrophs (arrowheads) in the vicinity of the follicles (F) are observed. Scale bars: A–F = 25 µm; Insets of all Figures = 5 µm.

Figure 6

Electron micrographs of pituitary pars distalis of adult male viscachas. (A–B) Conventional transmission electron microscopy. The folliculostellate cells (FSC) form follicles (F) have irregular nuclei (N), scarce cytoplasm with short cytoplasmic process (P) and absence of secretory granules. The different granulated cellular types (G) are closely associated with FSC, but without contacting the follicular lumen. Well developed junctional complex (arrows) are observed at the apical lateral surface of the FSC. These cells exhibit long microvilli (M) that protrude into the follicular lumen (F). (C–G) Transmission electron microscopy with lanthanum. (C) The tracer stops below the junctional complex (arrowheads) without penetrating in the follicular colloid. (D–F) Lanthanum freely penetrates into the intercellular spaces (arrowheads) between granulated endocrine cells (G) and FSC. The tracer does not penetrate into any healthy cellular type. On the contrary, lanthanum is observed inside the cells in different states of involution and degeneration (G). Scale bars: A = 7 µm; B–C = 4 µm; D–F = 3.33 µm; G = 2 µm.