Three-dimensional architecture and distribution of collagen components in the goat hypophysis.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 277A:275–286 (2004) Three-Dimensional Architecture and Distribution of Collagen Components in the Goat Hypophysis SHOTARO NISHIMURA,1* SHOJI TABATA,1 YOSHI-NORI NAKAMURA,1 KAORU OKANO,2 AND HISAO IWAMOTO1 1 Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka-shi, Japan 2 Department of Plant Resources, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka-shi, Japan ABSTRACT The three-dimensional architecture of collagen ﬁbrils in the connective tissue framework and the distribution of collagen types in the goat hypophysis were studied by the cell maceration method in combination with scanning electron microscopy (SEM) and immunohistochemistry. The pars distalis of the adenohypophysis consisted of many cell clusters. SEM revealed that the wall of cell clusters appeared as various-sized ﬂat bundles of collagen ﬁbrils woven in a basket-like conﬁguration. In the pars tuberalis, the aggregates of collagen ﬁbrils were denser and bundles thicker compared to the pars distalis. The density of collagen ﬁbrils changed from the pars tuberalis to pars distalis without a distinct border. The collagen framework in the pars intermedia was mainly divided into three parts, the dorsal region with large hollows, the middle region, and the ventral sheet facing the cavum hypophysis. In the lobus nervosus of the neurohypophysis, the collagen network exhibited a sponge-like appearance at low magniﬁcation. Collagen ﬁbrils of various sizes consisted of loose wavy bundles distributed around the cavities. Immunohistochemistry revealed types I, III, IV, V, and VI collagen throughout the hypophysis. It is concluded that to maintain structural and functional integration, the components of collagen are in different conﬁgurations throughout the regions of the goat hypophysis. Anat Rec Part A 277A:275–286, 2004. © 2004 Wiley-Liss, Inc. Key words: collagen; hypophysis; scanning electron microscopy; immunohistochemistry; goat Connective tissue maintains the structure and function in a diverse range of organs and tissues. Collagen is an important connective tissue component and interest surrounds the three-dimensional construction of the collagen network in organs. In endocrine organs, the three-dimensional architecture of the collagen framework has been shown in the adrenal gland (Kikuta et al., 1991) and thyroid gland (Morita et al., 1994) using a combination of the cell maceration method and scanning electron microscopy (SEM). It is well known that the adenohypophysis is composed of several types of endocrine cells and that cell aggregation forms cell cords or clusters surrounded by connective tissue components. Although there are many studies about adenohypophyseal cells, there are few about the connective tissue or extracellular matrix components in the adenohypophysis. Even those studies have mainly focused on the basement membrane components such as type IV collagen, laminin, ﬁbronectin, heparan-sulfate © 2004 WILEY-LISS, INC. proteoglycan, or entactin (Tougard et al., 1985; Vila-Porcile et al., 1987, 1992a, 1992b; Farnoud et al., 1992, 1994; Horacek et al., 1993; Murray et al., 1997). Only a few reports have described the distribution and architecture of other members of the collagen family in the adenohypophysis (Kaidzu et al., 2000). Here, we undertook a threedimensional study of the conﬁguration of collagen ﬁbers in the hypophysis. *Correspondence to: Shotaro Nishimura, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka-shi 812-8581, Japan. Fax: ⫹81-92-642-2942. E-mail: email@example.com Received 8 September 2003; Accepted 28 December 2003 DOI 10.1002/ar.a.20014 276 NISHIMURA ET AL. As in many animal species, the goat adenohypophysis is characterized by anastomosing cell clusters and connective tissue ﬁbers among the cell clusters (Khatra and Nanda, 1981). The aim of this study was to elucidate the characteristics of the three-dimensional architecture and distribution of collagen types in the pars distalis, pars tuberalis, pars intermedia, and lobus nervosus of the goat hypophysis by SEM in conjunction with the cell maceration method and immunohistochemistry. MATERIALS AND METHODS Animals Adult Tokara goats (Japanese native breed, 7 males and 12 females) were sacriﬁced by exsanguinations from carotid artery under deep anesthesia with pentobarbitone. The hypophysis was quickly removed and cut in the sagittal or frontal plane. General Histology For observation of the general structure, parafﬁn sections were prepared and classical dye staining was performed. The tissue blocks were ﬁxed with sublimate-formalin (saturated HgCl2:formalin, 9:1) for 1 day at room temperature and subsequently rinsed with tap water. The tissue block was then immersed in an iodine/70% ethanol solution for the removal of sublimate precipitate. Sections were then dehydrated through an ethanol series and conventionally embedded in paraplast. Sections of 4 m in thickness were cut, deparafﬁnized with xylene, and rehydrated through an ethanol series. The trichrome staining method described by Goldberg and Chaikoff (1952) was employed with a slight modiﬁcation. Scanning Electron Microscopy To observe the three-dimensional arrangement of collagen ﬁbers, the maceration method was applied as previously described by Ohtani (1987) with a slight modiﬁcation. After ﬁxation with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2– 4 days at 4°C, the tissue was rinsed with phosphate buffer and macerated in a 7.4% NaOH solution for 4 days at 25°C. The NaOH solution was exchanged twice a day. The tissue was then rinsed with distilled water for 3 days and treated with 1% tannic acid for 2 hr followed by 1% osmium tetroxide in a cacodylate buffer (pH 7.4) for 2 hr at 4°C. The tissue was subsequently dehydrated through an ethanol series and displaced in 2-methyl-2-propanol. Tissue was freeze-dried by use of Tis-U-Dry Freeze-Dryer (FTS Systems). The dried specimen was mounted on an aluminum holder and coated with Au using an Ion Sputter IB-3 (Eiko Engineering, Japan). Tissue sections were then observed by a Superscan SS-550 scanning electron microscope (Shimadzu, Japan) at the Center of Advanced Instrumental Analysis, Kyushu University. Immunohistochemistry Pieces of tissue were frozen in liquid nitrogen for immunohistochemical study of the distribution of collagen types. Tissue sections of 10 m in thickness were cut by a Leica CM1850 cryostat (Leica Instruments, Germany), then treated with acetone (5°C, 5 min) and 1% triton X-100 in PBS (pH 7.4) for 5 min. The ABC method was employed for immunohistochemistry using a Vectastain ABC kit (Vector Laboratories) in accordance with the attached protocol. Polyclonal antibovine type I, III, IV, V, and VI collagen antisera (LSL, Japan) were used at a working dilution of 1:1,000. The speciﬁcity of the immunostaining was conﬁrmed by omission of the primary antiserum. RESULTS Pars Distalis of Adenohypophysis The pars distalis of adenohypophysis was composed of many cell clusters. The clusters packed several to dozens of glandular cells and were irregular in shape and of varying size (Fig. 1A). Cell clusters were enclosed with connective tissue components stained with aniline blue using trichrome staining. Sinusoids distributed in the interspace of cell clusters did not penetrate into the clusters. The zona tuberalis, which was distinguished from other regions of the pars distalis by the predominant basophils, was observed in the rostroventral region of the gland (data not shown). No gender difference in the connective tissue proﬁle was apparent in this preparation. NaOH maceration retained no cell components for SEM. The collagen framework was seen throughout the gland. At low magniﬁcation, a cell cluster was evident as a basket-like compartment (Fig. 1B). The residual space of glandular cells in a cell cluster was continuous and their border was not always obvious because of the lack or complete absence of collagen septa in the cell cluster (Fig. 1C). At a high magniﬁcation, dozens of collagen ﬁbrils could be observed as made up out of various-sized ﬂat belt-like bundles. The bundles intersected each other randomly (Fig. 1D). A ﬁne and irregular collagen ﬁbril net covered the inner surface of the basket. In the pars distalis, a thick layer of connective tissue lay between large blood vessels and cell clusters (Fig. 2A, arrowheads). The wall of the large blood vessel was reinforced with a tough and continuous sheet of collagen with a rugged inner surface (Fig. 2B). In this collagen sheet, a thin net-like conﬁguration of ﬁbrils in the innermost layer covered the underlying layer of thicker bundles in a predominantly circular direction (Fig. 2C and D). The rugged surface was attributed to variation in bundle thickness, being dense and tough in some places and loose and thin in others (Fig. 2D). In comparison, sinusoids lay among cell clusters and shared the collagen wall with neighboring cell clusters (Fig. 2E). Fine solitary ﬁbrils were entwined on the inner surface of the wall like the large vessel, although the density was relatively less than in the latter (Fig. 2F). The ventral capsule of the gland, part of the dura mater, consisted of a stack of many collagen sheets in parallel to the gland surface (Fig. 3A). In each sheet, components of the sheet were formed from collagen ﬁbrils in a predominantly transverse arrangement and dense aggregation of collagen bundles (Fig. 3B). Light microscopy showed that the marginal layer facing the cavum hypophysis was lined with a single cuboidal or columnar layer of epithelium on a connective tissue layer (Fig. 4A). Under low-magniﬁcation SEM, the underlying collagen sheet was seen as a ﬂat plate of densely packed collagen ﬁbrils (Fig. 4B and C). In the collagen plate, the delicate surface appeared as a cloth of collagen ﬁbrils and thin bundles (Fig. 4D and E). A tough supporting layer was composed of thick collagen bundles crossing over each other (Fig. 4B). COLLAGEN ARCHITECTURE OF GOAT HYPOPHYSIS Fig. 1. Light and scanning electron micrographs of the pars distalis in the goat adenohypophysis. A: Light micrograph of pars distalis using trichrome staining. Sinusoids (arrowheads) are distributed among cell clusters (asterisks) in the surrounding connective tissue. Scale bar ⫽ 50 m. B: Scanning electron micrographs of the pars distalis. The wall of cell cluster is seen as aggregations of basket-like collagen ﬁbrillar bun- 277 dles. Scale bar ⫽ 50 m. C: High magniﬁcation of the white frame in B. Collagen ﬁbrils are mainly composed of the wall of each cell cluster (asterisk) and there are few inside. Scale bar ⫽ 10 m. D: Larger magniﬁcation of the white frame in C. Single or a few collagen ﬁbrils are randomly seen on the surface of the belt-like bundles. Scale bar ⫽ 2 m. 278 NISHIMURA ET AL. Fig. 2. Light and scanning electron micrographs of a blood vessel (BV) in the pars distalis. A: Light micrograph of a large blood vessel and surrounding parenchymal tissue using trichrome staining. Connective tissue layer (arrowheads) intervenes between BV and cell clusters (asterisks). Scale bar ⫽ 50 m. B: Scanning electron micrograph of the blood vessel and surrounding collagen framework. Scale bar ⫽ 20 m. C: High magniﬁcation of the surface of the BV in B. Collagen bundles were wavy and circularly coursed along the longitudinal axis. Scale bar ⫽ 5 m. D: High magniﬁcation of the surface of large blood vessel. Collagen ﬁbrils are randomly entwined like a net in the bundle surface. Scale bar ⫽ 2 m. E: The vestige of sinusoids (arrowheads) among cell clusters. The sinusoid is usually adjacent to cell clusters. Scale bar ⫽ 50 m. F: High magniﬁcation of the white frame in E. Scale bar ⫽ 5 m. COLLAGEN ARCHITECTURE OF GOAT HYPOPHYSIS 279 Fig. 3. Ventral capsule (VC) of the pars distalis (PD). A: Sagittal section of the VC shows it is composed of many layers of collagen sheets. Scale bar ⫽ 100 m. B: High magniﬁcation of the white frame in A. Most of the ﬁbrils are arranged circularly along the longitudinal axis of the gland. Scale bar ⫽ 20 m. Pars Tuberalis of Adenohypophysis The pars tuberalis is situated in the cranial region of adenohypophysis close to the median eminence. Histological sections showed that cell clusters were relatively small compared to those of the pars distalis (Fig. 5B). SEM showed that the smaller hollow with no glandular cells in the pars tuberalis was surrounded by a thicker wall of densely packed collagen bundles compared with the pars distalis (Fig. 5A). These features were also observed in the interclusteral band of connective tissue stained with aniline blue (Fig. 5B). This thicker wall of collagen bundles was predominant in the proximal region, where varioussized bundles intermingled with a random strand (Fig. 5C and D). However, a distinct border with a special conﬁguration of collagen bundles could not be determined between the pars tuberalis and pars distalis. Pars Intermedia of Adenohypophysis In histological specimens, the pars intermedia was in contact with the dorsal neurohypophysis through an intervening connective tissue layer, with branches penetrating the pars intermedia as septa carrying blood vessels (Fig. 6A). SEM showed the collagen framework of the pars intermedia had a rough dorsal region with large hollows, a middle region with many branches, and a ventral sheet facing the cavum hypophysis (Fig. 6B). In the dorsal region, ﬂat collagen sheets separated the gland into many compartments or lobules with few branches of collagen ﬁbrils or bundles. In the middle region, many branches of ﬁbrils and bundles from the collagen sheets made up a loose collagen mesh (Fig. 6C). The collagen sheet was composed of intermingled collagen ﬁbrils and bundles and some circular strands (Fig. 6D and E). The ventral collagen sheet in the pars intermedia over the cavum hypophysis had many pores in contrast to the opposite sheet of the pars distalis (Fig. 6B, arrowheads). Lobus Nervosus of Neurohypophysis The neurohypophysis was placed on the dorsal side of the adenohypophysis as described for the pars intermedia. Histological section in the sagittal plane showed that the connective tissue of the lobus nervosus was wavy and ran along the nerve ﬁbers and encircled nerve ﬁbers in the frontal section (Fig. 7A and B). Blood vessels were usually accompanied by thick connective tissues. Low-magniﬁcation SEM showed the collagen network in the lobus nervosus had a sponge-like appearance with felt-like collagen platelets and bundles enclosing various-sized cavities (Fig. 7C). The felt-like platelets of the collagen ﬁbrils were observed in many places and thin collagen bundles rose to distribute around cavities (Fig. 7D). Wavy or spiral collagen bundles were very common. Fibrils and bundles of various sizes intermingled with each other to form a loose network (Fig. 7E). The course of capillary vessels were not obvious in the SEM specimen. 280 NISHIMURA ET AL. Fig. 4. Light and scanning electron micrographs of the marginal layer of PD facing the cavum hypophysis (CH). A: Sagittal section of PD and pars intermedia (PI). The marginal layer of PD is composed of a single cuboidal or columnar epithelium and lining connective tissue layer. Scale bar ⫽ 50 m. B: Scanning electron micrographs of the connective tissue layer lining the marginal layer. High magniﬁcation of the white frame in C. The connective tissue layer is composed of a few layers of thick bundles of collagen ﬁbrils. Scale bar ⫽ 4 m. C: Low magniﬁcation of the surface of the marginal collagen layer. Scale bar ⫽ 50 m. D and E: High magniﬁcations of the surface of the layer. Scale bars ⫽ 100 m and 2 m, respectively. Immunohistochemistry brane. Immunolabeling by other antisera was detected in the remaining regions of connective tissue in the hypophysis. The distribution of all collagen types except for type IV was very similar and we could not distinguish speciﬁc ﬁbrils. Type I, III, IV, V, and VI collagen antisera labeled the connective tissue compartments of all regions of the adenohypophysis and neurohypophysis (Fig. 8). Here, only type IV collagen antiserum labeled the basement mem- COLLAGEN ARCHITECTURE OF GOAT HYPOPHYSIS 281 Fig. 5. Light and scanning electron micrographs of the pars tuberalis (PT) in the goat adenohypophysis. A: Scanning electron micrograph of PT in the sagittal plane. In the PT, the aggregation of collagen ﬁbrils is denser and thicker than in PD. Scale bar ⫽ 100 m. B: Light micrograph of the sagittal section of PT with trichrome staining. Cell clusters in this region are relatively smaller in size and there are more connective tissue components compared to the PD (see Fig. 1A). Scale bar ⫽ 50 m. C: High magniﬁcation of a part of the PT in A shows dense aggregations of collagen ﬁbrils. Scale bar ⫽ 40 m. D: High magniﬁcation of the white frame in C. Scale bar ⫽ 4 m. DISCUSSION three-dimensional organization of the reticular ﬁbers in the human pancreas (Ohtani, 1987). Although collagen frameworks in many tissues have been studied using this The maceration method with NaOH solution was ﬁrst developed for use in conjunction with SEM to image the 282 NISHIMURA ET AL. Fig. 6. Light and scanning electron micrographs of PI in the goat adenohypophysis. A: Light micrograph in the sagittal section shows the PI is separated from the lobus nervosus (LN) by an intervening connective tissue layer (arrowheads) on the dorsal side. B: Scanning electron micrograph of the PI in the sagittal plane. Many holes were observed in the ventral sheet (arrowheads). Scale bar ⫽ 500 m. C: High magniﬁ- cation of the white frame in B. Elongated ﬂat collagen sheets (arrows) project downward from the dorsal sheet and thinner bundles (arrowheads) and irregularly branch from the frames. Scale bar ⫽ 50 m. D and E: Higher magniﬁcations of a collagen sheet in the PI. The frames are composed of irregularly coursed collagen ﬁbrils. Scale bar ⫽ 4 m and 2 m, respectively. COLLAGEN ARCHITECTURE OF GOAT HYPOPHYSIS Fig. 7. Light and scanning electron micrographs of the lobus nervosus in the goat neurohypophysis. A and B: Light micrographs in the sagittal (A) and frontal (B) plane. Sagittal section shows that the connective tissue is wavy (arrowheads in A) and surrounds some nerve ﬁbers in the frontal section (arrowheads in B). Scale bar ⫽ 50 m. C: Low- 283 magniﬁcation scanning electron micrograph of the lobus nervosus. Scale bar ⫽ 100 m. D: High magniﬁcation of the lobus nervosus in C. Scale bar ⫽ 20 m. E: High magniﬁcation of the white frame in D. Scale bar ⫽ 4 m. 284 NISHIMURA ET AL. Fig. 8. Light micrographs of the goat hypophysis immunolabeled with anticollagen types I (A–C), III (D–F), IV (G–I), V (J–L), and VI (M–O). A, D, G, J, and M show labeling in the pars distalis. B, E, H, K, and N show labeling in the pars intermedia. C, F, I, L, and O show labeling in the lobus nervosus. Scale bar ⫽ 30 m. method, the three-dimensional architecture of collagen has only been reported in the endocrine organs of the adrenal gland (Kikuta et al., 1991) and thyroid gland (Morita et al., 1994). Here, we present the ﬁrst data on the three-dimensional architecture of collagen ﬁbers in the hypophysis. The pars distalis is the aggregation of cell clusters composed of glandular cells enclosed by connective tissue. Some organs, such as the adrenal gland and liver, also have cell clusters as their components. Although the adrenal gland has cell clusters in the zona glomerulosa, zona fasciculata, and zona reticularis of the adrenal cortex and adrenal medulla, the architecture of the collagen framework around cell clusters changes according to cord structure (Kikuta et al., 1991). The collagen architecture of the pars distalis shown in the present study is similar to that of adrenal medulla. In the liver, the architecture of the wall of space occupied by hepatocytes (Ohtani, 1988) is similar to that in the pars distalis seen here, although the residual space of hepatocytes is not basket-like but tubelike. In comparison, the thyroid gland has follicles lined with a simple cuboidal epithelium. The follicles are surrounded by perifollicular collagen sheaths composed of both thick collagen bands that run in various directions COLLAGEN ARCHITECTURE OF GOAT HYPOPHYSIS and ﬁne solitary collagen ﬁbrils (Morita et al., 1994). It also has an architecture similar to that found in the pars distalis. From these results, the basket-like architecture of the collagen framework is common in holding glandular cells in various endocrine organs. Sinusoids are generally distributed according to the endocrine function of the gland. SEM revealed that the hepatic sinusoid is surrounded with a wall of intermingled collagen and the collagen meshwork lining the hepatic sinusoids is characterized with many switchback ﬁbrils in human and rat liver (Ohtani, 1988). On the other hand, such switchback pattern of ﬁbrils were not so predominant in the collagen meshwork for adenohypophyseal blood vessels or sinusoids in this study. As hepatic sinusoids also have structural relationship with the spaces of Disse (Ohtani, 1988) but there are no such spaces in the adenohypophysis, the difference in the collagen meshwork for sinusoids may reﬂect the structure and function of liver and adenohypophysis. In the pars distalis, the density of solitary ﬁbrils surrounding the inner surface of vessels seemed to change according to the size, namely, they are denser in the large vessel and are sparser in the small vessels or sinusoids (Fig. 2D and F). It may be related to the structural strength of vessels. The pars tuberalis occupies the cranial region of the gland, to some extent enclose the infundibulum, and has an important role in connecting the pars distalis to the hypothalamus. It also strengthens the tissue by increasing collagen levels. In this study, SEM revealed the reinforcement of the pars tuberalis with many collagen ﬁbrils and that this tough architecture may have a greater effect on the smaller cell clusters compared to those in the pars distalis. The collagen sheets surrounding the large parenchyma in the pars intermedia had a unusual structure and were different from any other in hypophyseal regions. Although the compartment in the dorsal region contained either no or only a few collagen branches, the parenchyma in the middle region appeared to be supported by a coarse network of collagen branches. As the cavum hypophysis intervenes between the pars intermedia and pars distalis and the ventral surface of pars intermedia is free from pars distalis except for the lateral periphery in the goat hypophysis (Khatra and Nanda, 1981), many more collagen branches strengthen the structure of the middle region in the pars intermedia compared to the dorsal region. Interestingly, the architecture of the surface sheet of collagen in the side of the cavum hypophysis was different in the pars distalis and pars intermedia. The surface in the former was uniformly ﬂat while the latter had scattered pores. As there is a layer of epithelium on this surface in the intact organ, it indicates that epithelial cells in some regions on the side of the pars intermedia are directly attached to glandular cells in the pars intermedia. The signiﬁcance of this structure remains to be determined. The neurohypophysis is composed of neural tissue extending from the hypothalamus. As far as we know, there are no reports describing the three-dimensional architecture of collagen in the central nervous system. The present study showed that the architecture of the collagen network in the lobus nervosus of the neurohypophysis had a sponge-like conﬁguration and that collagen bundles had a wavy or spiral proﬁle. This architecture appears to be 285 more ﬂexible compared to that found in the cell cluster of the pars distalis and may determine the function of neural tissue. Although positive immunolabeling was detected throughout the goat hypophysis with type I, III, IV, V, and VI collagen antisera, we could not identify the type of individual collagen ﬁbrils in the macerated specimen either morphologically or immunohistochemically. As the maceration method removes all of the cellular elements, including their basal lamina (Ohtani, 1988), the type IV collagen component of the basal lamina may not reside in the macerated specimen. Thus, the macerated hypophyseal specimen consisted of at least four other types of collagens. An appropriate method is needed to clarify the distribution of each collagen type in the macerated specimen. Despite reports that show tumor GH3B6 prolactin cells produce basement membrane components in vitro (de Carvalho et al., 2000) and primary cultured adenohypophyseal cells in rat synthesize types I and III collagens (Kaidzu et al., 2000), the synthesis and secretion of collagen components by adenohypophyseal cells have not been demonstrated in vivo. The present study showed an absence of immunolabeling for any type of collagen in the adenohypophyseal cells. Kaidzu et al. (2000) suggested that collagen turnover in the adenohypophysis was slow in vivo and that the cells might monitor their environment and levels of collagen. During synthesis, collagen is modiﬁed from a large-molecular-weight precursor into procollagen, which has additional nonhelical polypeptide domains, before and after secretion (Davidson and Berg, 1981). Therefore, it may be available to detect the procollagen molecule or its mRNA for the identiﬁcation of collagen-producing cells. 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