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真珠袋形成に関する組織学的研究
https://fra.repo.nii.ac.jp/records/2009048
https://fra.repo.nii.ac.jp/records/2009048ebd6ff36-1f8a-43bf-abcf-253a84360518
| Item type | 紀要論文 / Departmental Bulletin Paper(1) | |||||
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| 公開日 | 2024-06-27 | |||||
| タイトル | ||||||
| タイトル | 真珠袋形成に関する組織学的研究 | |||||
| 言語 | ja | |||||
| タイトル | ||||||
| タイトル | Histological studies on the pearl-sac formation | |||||
| 言語 | en | |||||
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| 言語 | jpn | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_6501 | |||||
| 資源タイプ | departmental bulletin paper | |||||
| アクセス権 | ||||||
| アクセス権 | metadata only access | |||||
| アクセス権URI | http://purl.org/coar/access_right/c_14cb | |||||
| 著者 |
町井, 昭
× 町井, 昭 |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | （要約） 外套膜片移植手術（挿核手術）後，移植片の外側（殻側）上皮細胞が増殖遊出して挿入核面あるいは手術によって生じた宿主組織の傷面をおおう真珠袋を形成する過程をつぎの手段により追究した。 1) 生殖腺中に挿核手術したものならびに外套膜組織中に移植した外套膜片（イケチョウガイ）における真珠袋形成の組織切片による断面からの観察。 2) カバーグラス法―２枚のカバーグラスの間に移植片をはさんで宿主組織（閉殻筋）中に挿入し，一定期間経過後これを取り出して観察する方法―による生体観察ならびに固定染色標本による上面からの観察。 3) 真珠袋の第二次移植。 4) 外套膜の組織培養。 以上の方法により真珠袋形成過程を総合的に検討した結果つぎの結論を得た。すなわち，真珠袋を形成するのは外套膜の外側上皮細胞で，移植後移植片の外側上皮細胞は増殖してシート状に集まり移植片から遊出し，宿主の組織の傷面または傷面に現れた遊走細胞を足場にして核面を包むごとく真珠袋を形成する。そしてこの間において顆粒遊走細胞および無顆粒遊走細胞も増殖し，後者は真珠袋上皮細胞下と宿主組織の間あるいは移植片と宿主細胞との間において網目様に結びついて両者の癒合にあずかる。細胞の増殖は無糸核分裂によるのではないかということが推定された。真珠成分には有機質，稜柱層，真珠層の３成分がみられたが，これらを生成しているときの真珠袋上皮細胞の形態と染色態度にはそれぞれつぎのような特徴がみられる。 真珠層を生成している真珠袋では上皮細胞は一般にへん平で，ヘマトキシリン－エオジン染色では真珠層を生成している真珠袋上皮細胞と同じくヘマトキシリンに染まっているが，細胞質中にはエオジン好性の小さい顆粒が真珠層を生成している上皮細胞におけるよりも多く分布している。また上皮細胞間には，ヘマトキシリンによって塊状ないしは網目状に染まる内容物を含んだ粘液細胞が分布しているが，このような諸成分が分泌されて稜柱基質を形成する成分にくみ入れられることが推定される。 有機質を生成しているときの上皮細胞は円柱状のものが普通であるが，10μ くらいのあまり厚くないものからも有機質が生成されていることがある。上皮細胞中には顆粒成分が非常に多く，またエオジンに染まる大きな蛋白顆粒と思われる顆粒が充満した腺細胞が上皮細胞の各所に多数みられることがあり，これらの成分も分泌されて有機質の成分になるのではないかと推定される。 |
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| 言語 | ja | |||||
| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | In the last fifteen years, we have had Kawakami (1952, '53, '54), Ojima and Watanabe (1953), Aoki (1956), Machii and Nakahara (1957), Nakahara and Machii (1957), Tsujii (1960), Fujioka and Tateishi (1960, '61) reporting on the results of their histological studies of the pearl-sac formation in Japanese pearl oysters. In those studies, the behaviour, property and function of the epithelial cells of the explant as well as the period required for the performance of pearl-sac epithelium were discussed in detail. As regards the cell multiplication in the explant, however, only the proliferation of the epithelial cell and connective tissue cell are briefly mentioned by Ojima et al., and none of them have refered to the figure of the nuclear division. It is still remains unknown whether the cell proliferation may occur in the course of the pearl-sac formation, or simply the inserted nucleus is enclosed in by the migration of the outer epithelial cells of the explant. In the researches of the non-aseptic tissue culture of the molluscan mantle, Gattenby and Hill (1934) emphatically stated that the amitosis was observed existing in the amoebocytes forming a net work on the outgrowth of the explant. Frandre and Vago (1963) also reported that a number of ciliated epithelial cells of the mantle as well as lots of large spherical cells and fibroblasts, were obtained in the numerous sub-cultures accompanied by the continuous mitosis, through the plasma clot method. A couple years ago, Kenny (1964) reported on the chemically defined medium for the heart cell of the American oyster, Crassostrea virginica, and as to the nuclear division, he did support mitosis. On the contrary, Li et al. (1966) pointed out the presence of amitosis which can be recognized in the proliferation of the in-vitro cultured oyster heart cells. Perkins and Menzel (1964) reported that all the cells forming a reticulated monolayer cell sheet in the outgrowth of the explant should have their origin in the explant, because they could not gather any evidence of mitosis in their tissue culture of the mantle of the oyster, Crassostrea virginica. From the results of these researches, we can get at least two different opinions as to the cell proliferation, one upholding mitosis and the other amitosis. On the foundation of the former histological viewpoints and through the organic combination research of the results of the recent observations in living cells, the squash staining preparation obtained by the coverslip method (Wada 1961, '62) and the later results of the tissue culture of the mantle tissue, the author made more basic researches on the pearl-sac formation. The present study is therefore, to report on the process of the pearl-sac formation in the common pearl oyster, Pinctada fucata (Gould) and Schlegel's fresh water mussel, Hyriopsis (Limnoscapha) schlegelii (v. Martens), and especially to detail the particulars of the proliferation, arrangement and property of the cells derived from the grafted mantle tissue as the particulars of the morphology, behaviour and property of the wandering cells appearing on the operated cavity of the host's tissue or explant, during the early period of pearl-sac formation. The methods and techniques for the investigations are as follows. 1) Histological observations on the cross section of the grafted mantle tissue which is inserted into the gonad tissue with paraffin nucleus by the technique usually adopted in the pearl culture in the P. fucata and the observation of the mantle tissue transplanted into the connective tissue of the mantle of the Schlegel's fresh water mussel, H. schlegelii. After the operation for the transplantation of the small piece of mantle (nuclear insertion operation), specimens were collected every other day for the first 10 days, and after that every 3 to 5 days. Preparations were made by the commonest histological method. Specimens were embedded in paraffin and sectioned into 5 to 7 microns in thickness after being fixed in buffered 10% formalin (buffered at pH 7.0 by Na2HPO,, KH2PO.), acetic Zenker's and Ciaccio's fluid. And then followed the staining, Ehrlich's haematoxylin-eosin, Heidenhein's azan and Heidenhein's iron-haematoxylin staining, with those specimens. The fixatives, dates of fixation and staining methods carried out with these specimens are shown in Table 1. 2) The in-vivo culture of mantle tissue and normal pearl-sac. This is a coverslip method which is most suitable for the phase contrast microscopy, and for the observation of the cell proliferation and of the extent of cell migration by the squash staining preparation. In this method, a small piece of mantle or pearl-sac tissue, 1 to 2mm square in size, was interposed between two coverslips which had been cut in 5 to 8mm in size, and then inserted into the wound of the adductor muscle which had been cut open with a surgical knife. After that, the specimens were taken off from the adductor muscle every day or every other day for 20 days after the transplantation. Phase contrast microscopy (DM) was mainly attempted with the fresh preparation, while ordinary and polarizing microscopy were also made whenever necessary. Many of the coverslips taken off from the adductor muscle were treated with Ehrlich's haematoxylin-eosin, Rugaud's haematoxylin and Heidenhein's iron-haematoxylin staining. The Feulgen's reaction and the squash staining with 40% acetic orcein were also tested with some specimens. Buffered 10% formalin, 90% ethanol, Carnoy's fluid (3 : 1), acetic Zenker's and Rugaud's fluid were used as fixatives. The pre-fixation in 30% ethanol from one hour to night long which had been made prior to the above fixation resulted in a successful figure of the pseudopodia in fresh amoebocytes. Of these staining methods the acetic orcein squash preparation brought about the most distinct figure of the nuclear division. 3) The secondary transplantation of the pearl-sac tissue, in which the proliferation of the epithelial cells of the pearl-sac tissue were experimentally confirmed. The normal pearl-sac tissue consisting of a squamous epithelial cells which form a pearl layer, was used as explant. The explant 2 to 2.5mm square in size was inserted into the gonad of the mother oyster with paraffin nucleus 5 to 6mm in diameter. The specimens were treated by the common histological method which was mentioned above in 1). At the same time, the coverslip method was also adopted in this examination, in which the phase contrast microscopy was carried out with fresh coverslip which had been pulled out from the adductor muscle. 4) The in-vitro culture of the mantle tissue. In order to make clear the cell migration and arrangement in the early period of the pearl-sac formation, mantle tissue cut in 1 to 2mm square were cultured by the plasma clot method in which the chick embryo extracts were employed. In this method, Cameron's balanced salt solution for seawater squids and clams, which had been supplemented by the blood of pearl oyster, adductor muscle extracts and extracts from calf blood (Solcoseryl) was also used as liquid medium. Penicillin G and streptomycin were added as bacteriostatic agent (Table 2). The culture was carried out in TD-flask or modified Leighton tube at the room temperature, 18°C to 28°C. The phase contrast microscopy (DM) was used chiefly, and polarizing microscopy additionally. The whole aspect of the process of the pearl-sac formation, the cell migration, organization and proliferation have been made more clear through the above examinations. Here the relation of the shape and secretive property of the performed pearl-sac epithelium was concurrently discussed. Thus the present initial research in the tissue culture of the pearl oyster is expected to become a stepping-stone to the further advance in the research for the pearl formation. A number of wandering cells began to emerge from the explant and host's tissue after the nuclear insertion (Fig. 24). On the periphery of the explant, wandering cells which emerged from the explant and those from the host's tissue were in a mixed confusion. Two types of wandering cells, that is, granulocyte (granular leucocyte) and agranulocyte (lymphocyte, hyaline leucocyte reported by Takatsuki, 1934) were seen in the wandering cells which appear on the wound or the outgrowth of the explant. Granulocyte: This cell is active in movement, and is capable of expanding and contracting the pseudopodia in the host's tissue or in the culture medium. The spherical ones are 5 to 22 microns in diameter under the fresh condition. The cytoplasma contains bright spherical granules, 0.5 to 2 microns in diameter. The roundish nucleus has ten odd chromatin particles, and usually is maldistributed in one side of the cell, but nucleolus is invisible. The cytoplasma shows basophilic in nature in haematoxylin-eosin staining, and at times, some of the granulocytes contain a few eosinophile granules. In some cases, a number of wandering cells gather on the space formed between the surface of inserted nucleus and host's tissue. They are often found in the histological preparation of pearl and pearl-sac tissue to prove one of the causes which produce abnormal pearls. Agranulocyte: This is capable of amoeboid movement by the membranous pseudopodia, and shows a marked variation in shape. But the movement is not so prompt as granulocytes. A majority of these cells stretch the pseudopodia outwards in several directions (Figs. 25, 26, 48, 50, 51). Most of them are combined to each other with their complex pseudopodia, and these reticulated cell sheets, recognized as connective tissue by Gattenby and Hill (1934), Perkins and Menzel (1964), connect the newly formed pearl-sac epithelium to the underlying host's tissue or the wound which caused by operation, within 3 to some dozen days after the operation. The typical fibroblast-like cells with a most typically extended pseudopodia are distinctly seen in the in-vivo and in-vitro cultured cells, which, either attached to the coverslip or cutting into the plasma clot form a monolayer network. Cytoplasma is not so abundant as in granulocytes, but in the in-vitro cultured cells, many rough rod shaped twisted-thread-like or granular mitochondria are distinctly seen. The ovoid disc nucleus, relatively small in size, usually has ten odd chromatin granules under the phase contrast microscopy (DM). No active movement is observable in these reticulated fibroblast-like cells (Figs. 67, 68). Towards that period, a small number of ovoid or pear-like shaped outer epithelial cells also begin to emerge from the definite cut edge of the explant. Some of them, with spear-shaped cytoplasmic process, migrate in the space between the surface of the inserted nucleus and the host's tissue in their infirm wandering activities (Fig. 4). The cell sheet, which grew out of the definite cut edge of the explant, begins to extend outwards to enclose the surface of the inserted nucleus within one or two days after the operation (Fig.6). In summer, when the water temperature 28C to 30℃, it takes only 3 days for it to form the pearl-sac epithelium, but at lower water temperature, it requires longer time; for example, in April, 17 days, in September, 7 days. In these newly formed pearl-sac epithelia, marked variations in size and shape are observed with the portions. The epithelium formed on the portion where the explant once was, is usually columnar and has a property of forming an organic matter. The inner epithelial cells of the explant begin to degenerate several days after the operation, and most of them disappear in the course of the pearl-sac formation. It is interesting, however, that the explant, when the outer epithelium is experimentally cut off, sometimes forms a ciliated columnar epithelium which is derived from inner epithelium of the mantle (Figs. 12, 13, 14). But even in such cases, the epithelium is supposed to degenerate before long, which is easily surmised from the fact that in 35 day preparations only the wandering cells are surrounding the inserted nucleus (Fig. 15). As no pearl substances are found secreted from this epithelium, it is unquestionable that inner epithelium of the mantle has no functional ability to form a pearl-sac. The results of the secondary transplantation of the pearl-sac tissue forming a normal pearl layer, made it evident that the regenerated pearl-sac (secondary pearl-sac), as was seen in the pearl-sac epithelia derived from the primary transplantation of the mantle tissue, can be classified into three different categories according to their shape and secretive product. No difference can be recognized between the functions of these two pearl-sac epithelia. At the same time, however, the significant fact was experimentally proved that cell proliferation occur throughout the course of the pearl-sac formation. If the cell proliferation does not occur in the course of the pearl-sac formation, the epithelial cells in the regenerated pearl-sac must be either distinctly flat in shape, or in some cases, unable to cover all the surface of the inserted nucleus. However, on the newly formed secondary pearl-sac epithelium, as mentioned above, the epithelia which contain three different types of cells and cover all the surface of inserted nucleus are formed. In the present experiment, the same histological method as taken in 1) and the coverslip method mentioned in 2) were simultaneously employed together. In the 5 day preparation, the earliest specimens had already completed the secondary pearl-sac, and in 8 day preparation, a little prism had already been observed. The histological change was the same in the secondary transplantation as in the primary transplantation (Figs. 22, 23). Also, in the coverslip method in which a normal tissue of the pearl-sac was used as explant, numerous wandering cells appeared on the edge of the coverslip and in the surroundings of the explant, as did in case of the primary transplantation. From the surface of the pearl-sac epithelium, most of the squamous epithelial cells take polygonal in shape, each side extending to 5 to 25 microns in length. The outer edge of each cell is bordered with the relatively darkened narrow line. The cell nucleus shows variations, from 2 to 10 microns in diameter; larger ones extending more than 10 microns. Beneath the epithelial sheet, muscle fibres were observed fairly distinctly underlying in a striped pattern (Figs. 46, 47). In the surroundings of the definite cut edge of such an explant, round or ovoid epitheloid cells emerge from a monolayer cell sheet of pearl-sac epithelium. In the outgrowth of 4 day preparation, loosely arranged epitheloid cells were recognized (Figs. 48, 49). It was safely concluded, therefore, that in early October, the monolayer of the epitheloid cells has already performed a secondary pearl-sac which encloses the whole surface of the inserted nucleus or the whole two coverslips, within 10 days or so after the operation. The two day preparation in the coverslip method in which a mantle tissue was used, showed a significant figure of the cell proliferation in which the division of the supposed cytoplasma as either granulocyte or the epitheloid cell were observed in the fresh materials under the phase contrast microscopy (Fig. 35). In the 7 day preparations, it seemed that the coverslips were enclosed in with rough epithelial sheet, and some of them had already started to secrete an organic matter onto the coverslips (Fig. 41). During 11 days to 25 days after, a remarkable deposition of prism (spherulite of CaCO3) was observed continuously (Figs. 42-44). One or two months after the operation, the whole surface of the diplo-coverslips have got covered with pearl layer. It is safely concluded, therefore, that the outer epithelial cells of explant should have migrated onto the outside of the diplo-coverslips, according to their migratory activities (Text Fig. 1). As for the chromatic figure, ten odd chromatin granules were seen scattering about in the nucleus, and no other evidences for mitosis than the coarctate nucleus which reminds us of one type of the telophase (Figs. 52, 53, 56, 57). The nucleus in every epithelial cell, fibroblast like cell and granulocyte have nuclear membrane, and chromatin particles are clearly stained with 40% acetic orcein in squash preparation or haematoxylin staining. From the above observations on cytokinesis and nuclear division through the coverslip method and from the observation of the secondary transplantations of mantle tissue may possibly be obtained the following conclusions that after the transplantation of mantle tissue or pearl-sac, cell proliferation occurs in some epithelial cells and connective tissue cells of the explant and also in some wandering cells throughout the course of pearl-sac formation, and that amitosis might occur in these cells. The epithelia of pearl-sac may be classified into three categories in accordance with their own secretive properties, whether secreting an organic matter, forming a prismatic layer or forming a pearl layer. The squamous epithelium which forms a pearl layer is 2 to 10 microns in thickness, and has a basophilic nature, containing a few slender cytoplasmic granules. The nucleus is ovoid to ovoid disc in shape and contains one or two nucleoli and ten odd chromatin granules. Usually, the epithelial cells form relatively dense cytoplasmic layer on the free surface. Mucous cells are sometimes seen scattering here and there between the epithelial cells (Figs. 17, 18). In the decalcified preparations of the prismatic layer and of the epithelium which forms it, there is a thick eosinophile inter prismatic matrix vertically arranged to the epithelial layer, and in the matrix there is a fine haematophile inter lamellar matrix running at right angles. In contrast with the epithelia which forms a pearl layer, those of the prismatic layer are varied in shape and size, measured 5 to 30 microns in thickness, and some of them fill their cytoplasma with a great number of fine eosinophile granules which may be considered to turn into one of the elements of the prismatic layer. Nucleus is ovoid, or flat-ovoid relatively small, existing in the middle of the cell or between the middle of the cell and the basement membrane. Mucous cells are scattered between the columnar epithelial cells. They are basophilic in nature, dyeing violet slightly with haematoxylin-eosin staining (Fig. 19). The results of histological staining in the prismatic layer and it's epithelium may indicate that both the eosinophile granules contained in the epithelial cells and the haematophile substances which fills the cytoplasma of mucous cell may change into one of the elements of the prismatic substance. In comparison with the epithelial cell forming a pearl layer or a prismatic layer, the organic ones contain far more eosinophile granules which are considered to be proteinoid secretive granules in their cytoplasma. They are 10 to 50 microns in thickness. They sometimes take a form of the so-called pseudostratified ciliated or non-ciliated columnar epithelium, but most of them are ordinary simple columnar epithelia (Figs. 20, 21). Between columnar epithelial cells, mucous cells or unicellular glandular cells which are filled with large eosinophile granules are scattered. The free surface of the epithelial cells is usually rough in nature, and in some cells it is brushbordered or ciliated. But a few cells which are secreting a thread-like organic matter form a variated cytoplasmic process which may suggest the highly activated secretive function of these cells. According to the histological staining reaction, the organic pearl substance may be divided into the following three categories: 1) periostracum-like matter staining with eosin 2) organic matter staining red slightly with eosin and redish with azan staining 3) fibrous organic matter staining blue with azan staining. Doubtless all of them are secretive products from columnar epithelia, but still unknown is the cell or the epithelium which would secrete each of them. From the results of the observations in shape and staining reaction, it may safely be concluded that in comparison with the epithelium which is forming a prismatic or a pearl layer, the columnar one may actively support the formation of an organic matter in the secretion of protein. A few results from the tissue culture of mantle tissue are as follows: In the 4 hour preparation, roundish granulocyte, fibroblast like cell and epitheloid cell begin to emerge from the definite cut edge of the explant, extremely increasing in number for 1 to 2 days after that. In many of the 2 day cultures, round or roundish epitheloid cell sheets are formed on the outside of the explant, as if surrounding it. They are recognized to be a mass of dark round cells, and between the epitheloid cells, roundish cells which appear to be granulocyte with a somewhat irregular cytoplasma, are scattered (Fig. 59). In the 4 and over 4 day preparations, epitheloid cell sheets are extending further outwards, and in the periphery of this cell sheet, reticulated monolayer cell sheet which forms itself on the solid substrate or on the surface of coverslip is spreading out. Spindle and elongate shaped muscle cells which may be considered to be in the most typical shape in these cells also begin to appear on the edge of the explant. They are seen scattering singly or in bundle, half floating in liquid medium. In the phase contrast and ordinary microscopy, the internal structure could not be observed satisfactorily. It was clearly observed, however, that it had a dense bright muscle fibre along the perpendicular cell axis under the polarizing microscopy (Figs. 62-66). In the most cases, the culture could be kept on for more than 10 days. In some of the 10 to 20 day preparations, extremely flattened fibroblast-like cells were reticulated together by their distinctly stretching pseudopodia (Figs. 67, 68). The best ones were kept in culture for 45 days. | |||||
| 言語 | en | |||||
| 内容記述 | ||||||
| 内容記述タイプ | TableOfContents | |||||
| 内容記述 | （目次） １．緒言 ２．謝辞 ３．真珠袋形成の組織学的観察 ４．In-vivo における真珠袋形成の観察 ５．In-vitro における真珠袋形成の観察 ６．総括 ７．考察 ８．要約 ９．Summary 10．文献 |
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| 言語 | ja | |||||
| 書誌情報 |
ja : 国立真珠研究所報告 en : Bulletin of the National Pearl Research Laboratory 巻 13, p. 1489-1539, ページ数 51, 発行日 1968-07-05 |
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| 出版者 | ||||||
| 出版者 | 国立真珠研究所 | |||||
| 言語 | ja | |||||
| 出版者 | ||||||
| 出版者 | National Pearl Research Laboratory | |||||
| 言語 | en | |||||
| 書誌レコードID | ||||||
| 収録物識別子タイプ | NCID | |||||
| 収録物識別子 | AN00091717 | |||||
| 情報源 | ||||||
| 識別子タイプ | Local | |||||
| 関連識別子 | pearl_k_1489 | |||||
| 関連サイト | ||||||
| 識別子タイプ | URI | |||||
| 関連識別子 | https://jp-pearl.com/wp-content/uploads/2018/06/houkoku013.pdf#001 | |||||
| 言語 | ja | |||||
| 関連名称 | 日本真珠振興会Archive | |||||
