Arch. histol. jap., Vol. 50, No. 5 (1987) p. 567-578
Differentiation
of Striated
Muscle
Fibers
in Pituitary
Gland
Grafts
Transplanted
beneath
the Kidney
Capsule*
Kinji INOUE, Yutaka TANIGUCHI and Kazumasa KUROSUMI
Department of Morphology (Prof. K.KUROSUMI), Institute of Endocrinology, Gunma University, Gunma, Japan
Received October 1, 1987
Summary. Striated muscle fibers appear with regularity in pituitary isografts transplant-ed beneath the kidney capsule. These differentiated striated muscle fibers consist of multinucleated cells resembling skeletal muscle fibers. They appear mainly in the pars distalis, though occasionally in the gars intermedia of the grafts. Although most glandular cells disappeared due to necrosis in the center of the grafted anterior pituitary, where the striated muscle fibers occurred, the folliculo-stellate cells were well preserved in this region and formed elongated myotube-like structures. The muscle fibers appearing in the central pars distalis were in close contact to the folliculo-stellate cells and also to the marginal epithelium of the pituitary cleft. In these cases the striated muscle fibers and folliculo-stellate cells were surrounded by a common basal lamina. This close association between heterotopically differentiated muscle fibers and the normally occurring folliculo-stellate cells strongly suggests a close relationship in differentiation and function between these different cells.
Heterotopic striated muscle fibers have been observed in the pineal gland (DIEHL,1978), the brain (HoFFMAN and RORKE,1971; AMBLER, 1977; NAKAMURA et al., 1984) and the thymus (WEKERLE et al., 1975). The occurrence of these heterotopic striated muscle fibers attracts attention from both the embryological and clinico-pathological view-points. The occurrence of muscle fibers in the thymus is especially intriguing, since it has been suggested to be related to the pathogenesis of myasthenia gravis (PALESTRo et al., 1983).
Recently, BRUNNER and TSCHANK (1982) have described the appearance of striated muscle in the cultured anterior pituitary, which is a very aberrant location. Before this, the origin of this striated muscle, i.e., the cells from which it differentiates, had remained completely unknown. In the present study, we transplanted pituitaries heterologously beneath the kidney capsule to observe and study the resulting mor-phological changes occurring in the glandular cells when isolated from stimulation by hypothalamic releasing factors. During the course of the study, we unexpectedly observed that striated muscle fibers differentiated within the pituitary grafts. We here discuss the origin of these striated muscle fibers.
*This work was supported in part by a research grant from the Ministry of Education , Science, and Culture, Japan.
568 K. INOUE, Y. TANIGUCHI and K. KUROSUMI:
MATERIALS AND METHODS
Adult male rats of the Wistar strain weighing between 250 and 300 g were used in this study. The rats were maintained under a 14L :1OD photoperiod with food and water available ad libitum. Donor rats were sacrificed by decapitation. Immediately after sacrifice, the pituitaries which contained all the lobes (anterior, posterior and interme-diate) were carefully removed from their capsules. The kidney of the host animal was exposed through a dorsal surgical approach under anesthesia with pentobarbital (5 mg/100 g b. w., i.p.) with a deep pocket made beneath the kidney capsule. The donor pituitary was placed in this pocket, the posterior lobe side contacting the surface of the kidney. During surgery, great care was taken to avoid any injury to the donor pitui-taries. The host animals were maintained under the same conditions as those described above until fixation of the transplanted pituitaries. One to two weeks after transplanta-tion, the hosts were sacrificed by decapitation and the grafts fixed for light microscopic observation in a modified Bouin fixative (INouE and HAGINO, 1984), Specimens for electron microscopy taken from the other animals after the same treatment were cut into 1 mm3 cubed pieces of tissue and carried out through a double fixation with 2.5% glutaraldehyde and 1% osmium tetroxide in phosphate buffer by immersion fixation. After conventional dehydration, the grafts were embedded in either Paraplast for light
microscopy or Epon-Araldite for electron microscopy.
Immunocytochemistry of light microscopy was performed by the PAP method (STERNBERGER et al., 1970) for staining of the striated muscle fibers and folliculo-stellate cells using anti-human myoglobin (Cooper Biomedical, Pennsylvania, USA) or anti S-100 protein (Immuno-Biological Laboratory, Takasaki, Japan), respectively. The working dilution of these antibodies was 1 : 1500 for anti-serum of myoglobin and 1 : 1000 for anti-serum of S-100. Staining procedures have been described elsewhere (INOUE and HAGINO,1984). The specificity of the staining was proved by an absorption test or the substitution of the primary antibody with normal serum. No reaction was
observed in specimens treated with the above control staining.
RESULTS
Light microscopy
The peripheral region of each grafted pituitary appeared almost intact, showing the same morphology as that of the normal gland. However, the center of the grafted gland was necrotic and the usual glandular cells had almost disappeared by 10-14 days after transplantation. The striated muscle fibers were mainly present in these necrotic areas of the grafted pituitary (Fig. 1). Almost all of the pituitary grafts contained differentiated striated muscle fibers, although the number of muscle fibers varied considerably among grafts.
Differentiated muscle fibers were occasionally observed in the marginal layer of the pars intermedia (Fig. 2). Some of the differentiated muscle fibers were seen in close contact with the epithelial cells of the marginal layer facing the pituitary cleft (Fig. 3). The differentiated muscle fibers showed clear striations and contained many nuclei (Fig. 3) ; they were therefore regarded as skeletal muscle fibers. The muscle fibers were intensely immunostained with anti-human myoglobin serum. All of these
immuno-Striated Muscle Fibers in Pituitary (rafts 569
positive fibers were clearly shown to be striated on the myofibril patterns; no other kind of cells in the graft were stained with the anti-myoglobin serum.
The folliculo-stellate cells of the pars distalis and marginal layer cells of the pars intermedia were well stained by immunocytochemistry using anti-S-100 protein (Fig. 4).
Fig. 1. Immunohistochemical demonstration of myoglobin in striated muscle fibers appearing in a grafted pituitary beneath the kidney capsule. Many striated muscle fibers (arrowheads) appear in the center of the pars distalis of the graft. CA kidney capsule, AL anterior lobe, PC pituitary cleft, PIL posterior and intermediate lobe, K kidney parenchyma. X240
Fig. 2. Myoglobin-containing striated muscle fibers (arrowheads) in the marginal cells of the pars intermedia. IL intermediate lobe, AL
570 K. INOUE, Y. TANIGUCHI and K. KUROSUMI:
These staining characteristics with the antibody against S-100 protein in the grafted pituitary were almost the same as those in the intact glands except for the difficulty of staining folliculo-stellate cells in the central portion of the graft (Fig. 5). Immunocytochemically, S-100 protein was not observed in differentiated muscles.
Fig. 3. A multi-nucleated striated muscle fiber (MU) stained with the antibody to myoglobin. The muscle fiber is in close contact with marginal epithelial cells (arrows) facing the residual cavity of Rathke's pouch (PC).
x 1,500
Fig. 4. Immunocytochemical demonstration of S-100 protein in the normal male rat pituitary. Folliculo-stellate cells (arrowheads) in the pars distalis and marginal cells (MC) of the pars inter-media are positively stained. X500
Striated Muscle Fibers in Pituitary Grafts 571
Electron microscopy
As previously described with regard to our light microscopic observations, the charac-teristics of the outer region of each grafted pituitary were almost identical to those of the intact gland. As mentioned before, however, the center of the pituitary graft degenerated as most of its glandular cells disappeared. The folliculo-stellate cells in the external region of each graft showed typical morphological features such as the formation of cell clusters surrounding a small follicular lumen filled with an electron-lucent colloidal substance and extending extremely long cellular processes which
frequently embraced neighboring glandular cells (Fig. 6). Some of the folliculo-stellate cells in the center of the graft resembled those found in the normal peripheral region, where they possessed very long cellular processes and formed an electron lucent follicular lumen. However, their long cellular processes did not surround secretory cells, since the latter had already been lost through necrosis (Fig. 7). Other folliculo-stellate cells found in the center of the graft possessed a very long tubular structure containing collapsed follicles filled with an electron dense colloidal substance (Fig. 8). Furthermore, some elongated cells formed an extremely shrunken follicular lumen (Fig. 9). Their microvilli were closely packed and devoid of any colloidal substance. The cell borders of these extended folliculo-stellate cells tended to be indistinct, thereby forming a structure resembling a myotube (Fig. 9).
These tubular cells were frequently adjacent to well differentiated striated muscle fibers (Fig. 9, 10, 11). Where muscle cells and folliculo-stellate cells were in contact, they shared a common basal lamina (Fig. 10). Myofilament-like structures were ob-served in folliculo-stellate cells which were in close contact with striated muscle fibers (Fig. 11).
As described above, muscle fibers differentiated mainly in the centers of the
Fig. 5. Localization of S-100 protein in the pars distalis of the pituitary graft. The immunoreactive cells are seen in a peripheral part of the pars distalis (arrowheads). On the other hand, no immuno-positive cell is seen in the center of the pars distalis, where many degenerated cells are seen (asterisk). X 500
772 K. INon~G, Y. 'I AVAGucni and K. Kuiiosuxii.
Fig. 6. Electron micrograph of folliculo-stellate cells (FS) in an area near the surface of the anterior pituitary graft. Folliculo-stellate cells form an electron lucent follicular lumen (L) and long cellular processes of folliculo-stellate cells embrace normal looking glandular cells (GC). x3,000
Fig. 7. Foil iculo-stellate cells (FS) surround a dilated follicular lumen (L) situated in the deep portion of an anterior pituitary graft. Although the folliculo-stellate cells are well preserved, nearly all of the glandular cells have disappeared as a result of necrosis (asterisks). X3,000
Striated Muscle Fibers in Pituitary Grafts ;573
Fig. 8. Folliculo-stellate cells forming collapsed follicles filled with an electron dense colloi-dal substance (arrow). X 11,000
Fig. 9. A very elongate folliculo-stellate cell exihibiting an extremely compact follicle (arrow). Folliculo-stellate cells (FS) are intimately associated with a differentiated striated muscle fiber (MU). Arrowhead shows myofilaments. X4,000
574 K. INOUE, Y. TANIGUCHI and K. KuxosuMI:
pituitary grafts, where almost all of the glandular cells had disappeared. In a few cases, however, striated muscle cells were observed coexisting with secretory cells. In such cases, no basal lamina was found between the striated muscle cells and secretory cells (Fig. 12).
DISCUSSION
Occasional reports of the heterotopic striated muscle can be found in the literature. It has been confirmed in the thymus (WEKERLE et al., 1975), in the brain (HOFFMAN and RORKE, 1971; AMBLER, 1977; NAKAMURA et al., 1984) and in the pineal gland (DIEHL, 1978), under both normal and pathological conditions. On the other hand, skeletal muscles which, as is widely accepted, are usually originated from the mesoderm, have been proposed by DOUARIN (1982), to be partly derived from the neuroectoderm. The hypothesis of DOUARIN is supported by results from her skillful transplantation of a quail embryo neural crest into a chick embryo. After this experiment she demonstrated that migratory neural crest cells differentiated into mesenchymal elements including skeletal muscles in the chick embryo ; these mesenchymal elements originating from the neural crest were termed mesoectoderms. This relationship between the neural crest and striated muscles was also supported by studies using Thy-1 antigen which was first identified as a surface antigen of thymocytes and also demonstrated in myoblast cells or early stages of myotube as well as nervous elements including the astrocyte (BARCLAY et al., 1976). Furthermore, neuron specific enolase (NSE) was demonstrated in heterotopically appearing striated muscles and neighboring glial elements. Therefore, some of these heterotopic striated muscle fibers are thought to be of neuroectodermal origin (NAKAMURA et al., 1984). The neuroectoderm may have the
Fig. 10. The close association between folliculo-stellate cells (FS) and striated muscle cells (MU). They are surrounded by a common basal lamina, as indicated by arrows.
Striated Muscle Fibers in Pituitary Grafts 575
Fig. 11. a and b. Folliculo-stellate cells (FS) in close contact with striated muscle cells (MU) containing myofilaments. The folliculo-stellate cell also contains filaments in the cytoplasm (a). The area indicated by an arrow in a is enlarged in b, showing the myofilament-like structure. a: x23,000, b: x70,000
a
576 K. INOUF, Y. TANIGUCHI and K. KuROSCNHH
potential to differentiate into the striated muscle. The clearest demonstration of this phenomenon has been seen in the differentiation of a striated muscle from the cultured glial cell line known to be of neuroectodermal origin (LENNON et al., 1979). On the other hand, some of the cellular components of the anterior pituitary gland are suspected to originate from the neuroectoderm (TAKOR and PEARSE, 1975; PEARSE and TAKOR, 1979) or to be paraneurons (Fu.JITA, 1980). In particular, the folliculo-stellate cells in the anterior pituitary are strongly suspected to be a glia-like element, since they contain S-100 protein (NAKAJIMA et al., 1980, COCCHIA and MIANI, 1980) or glial-specific acidic protein (GFRP; VELASCO et al., 1982). If the folliculo-stellate cells in the pars distalis are indeed glia-like cells, they may have the potential to differentiate into the striated muscle fibers.
Thus far there has been only a single report, by BRUNNER and TSCHANK (1982), on striated muscle fibers differentiated from cultured anterior pituitary cells. These authors stated that this striated muscle might have differentiated from the pituitary elements, but not from migrating connective tissue elements. The result of our present study shows that striated muscle fibers obviously appear in the center of the grafted anterior pituitary and are surrounded by normal pituitary cells. Therefore, we also believe that this heterotopic striated muscle has been differentiated from cells
compos-ing the pars distalis. This findcompos-ing offers an alternate interpretation of the origin of the heterotopic striated muscle in transplanted pituitaries.
Our electron microscopic study showed that striated muscle fibers occasionally lay
Fig. 12. A well differentiated striated muscle (MU) and a prolactin cell (PL) of the anterior pituitary in close contact with each other without any basal lamina separating.
Striated Muscle Fibers in Pituitary Grafts 577
adjacent to folliculo-stellate cells, and we also found that these two cell groups were surrounded by a common basal lamina. On the other hand, ORDRONNEAU and PETRRUSz (1986) have immunocytochemically demonstrated the presence of desmin, one of the muscle-specific proteins, in the marginal cells of the pars intermedia, the marginal cells of pars distalis facing the pituitary cleft and also the stellate cells of the pars distalis. This distribution of desmin in the normal pituitary is probably closely related to the appearance of muscle fibers in the grafted anterior pituitary in this study. Therefore, it is suggested that the marginal cells of the pars distalis facing the pituitary cleft, marginal layer cells of the pars intermedia, and folliculo-stellate cells have the poten-tial to differentiate into striated muscle fibers under conditions of grafting or culture.
However, we demonstrated the presence of myoglobin only in well differentiated striated muscle fibers, and not in any other cellular components in the pituitary grafts. Furthermore, we showed that S-100 protein was present in folliculo-stellate cells, the peripheral layer of the lobules in the pars intermedia and in that part of the marginal cells of the pars distalis facing the pituitary cleft, but failed to demonstrate it in differentiated striated muscle fibers. This might be explained by assuming that after differentiating into the muscle, these cells completely lose their prior characteristics. However, we cannot neglect the possibility that the pituitary gland might contain a few myoblasts which may be stimulated to grow, once they have been transplanted to ectopic places. Another possibility is the migration of circulating host myoblasts (MCGEACHIE and GROUNDS, 1985). Further studies will be needed to decide which of these alternatives can be ruled out.
In spite of the fact that pituitary transplantation is a classical method for the study of pituitary function, to our knowledge, no report has mentioned the appearance of striated muscle fibers in pituitary grafts. One predisposing condition for the appear-ance of striated muscle may be necrosis in the graft. In the present study, whole donor pituitaries were carefully removed and transplanted under the kidney capsule. With this procedure, the center of each grafted pituitary showed marked necrotic change due to the lack of circulation. This apparently stimulated the differentiation of striated muscle fibers that we observed in this study.
Acknowledgements. The authors gratefully thank Dr. E. F. COUCH, Department of Biology, Texas Christian University, Fort Worth, Texas, U. S. A., for his kind reviewing of the manuscript.
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井 上 金 治 〒371前 橋市 昭和 町 群馬 大学 内分 泌研 究所 形態 学研 究部 Dr. Kinji INOUE Department of Morphology Institute of Endocrinology Gunma University Showamachi, Maebashi 371 Japan
