Fundamental Studies on in vivo and in vitro Pearl Formation—Contribution of Outer Epithelial Cells of Pearl Oyster Mantle and Pearl Sacs

Masahiko Awaji1* and Akira Machii2

1National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhama, Minami-Ise, Watarai, Mie 516-0193, Japan

21557-6 Tsuiji, Isobe, Shima, Mie 517-0218, Japan

Abstract

Outer epithelial cells, which constitute a monolayer epithelium covering the outer surface of pearl oyster mantle, play principal roles in shell and pearl formation. In pearl culture, a fragment of the mantle prepared from a donor is implanted into the recipient's gonad together with a small inorganic bead. Histological studies using pearl oyster Pinctada fucata have revealed that the outer epithelial cells emigrate from the allograft, proliferate, and form a pearl sac surrounding the bead. Following the pearl-sac formation, the pearl-sac epithelia start to form calcium carbonate crystals, such as nacre, on the bead showing morphological characteristics closely related with the crystal structures. To investigate cellular mechanisms of the pearl formation, organ and cell culture methods for the outer epithelial cells of pearl oyster mantle were developed. In the organ culture, crystal formation, deposition of shell matrix-like structure, and DNA synthesis of the outer epithelial cells were observed. The outer epithelial cells separated from the mantle started DNA synthesis in co-culture with hemocytes that revealed a part of cell-to-cell interactions during the pearl-sac formation processes. Substitution of the cultured outer epithelial cells for a mantle allograft in pearl culture was tested by injection of the cultured cells; the results of which implied future possibilities for the application of the cultured outer epithelial cells for pearl production.

Keywords

pearl, mantle, cell culture, organ culture, Pinctada, Mollusca


1. Introduction

Molluskan shell is produced by mantle tissue that lines the inner surface of the shell. The mantle produces species-specific shapes, colors, and luster, activities that are under the control of the endocrine systems (Wilbur and Saleuddin 1983). When a part of the shell is damaged, a mantle starts to regenerate the shell in cooperation with the immune system (Watabe 1983; Mount et al. 2004). The outer surface of a mantle is covered by a monolayer of epithelial cells, and these epithelial cells have principal roles in the production and regeneration of a shell. In the case of pearl oysters in the genus Pinctada, the mantle is divided into three zones from the ventral to dorsal side as follows: marginal zone, pallial zone, and a central part (Tsujii 1960). The boundary between a pallial zone and a central part is the pallial line, which is an impression of the pallial muscles lined in an arc in the middle of a mantle. Several types of epithelial cells are recognized in the outer epithelium of a pearl oyster mantle (Tsujii 1960; Wada 1966). The most abundant type is named as outer epithelial cells. They are regarded to play a major role in shell formation and regeneration, and their functions are a main theme of this article.

A shell of pearl oyster Pinctada fucata, characterized by its beautiful luster over the inner surface, consists of two layers: periostracum and ostracum. The periostracum is a thin, pliable proteinaceous sheet covering the outer surface of the shell. Components of the periostracum are synthesized in a periostracum gland located at the bottom of a periostracal groove between an outer and a middle fold of a mantle. The periostracum is thought to serve as a barrier that isolates the interface between a shell and a mantle (extrapallial space) from the outer environment, and as an initial matrix for the deposition of calcium carbonate crystals, namely the ostracum (Saleuddin and Petit 1983).

The ostracum of a pearl oyster shell consists of three layers: outer, middle, and inner shell layers (Wada 1957). The outer shell layer is composed of calcite crystals organized in a prismatic structure, therefore referred to as a prismatic layer. The inner and middle layers are aragonite crystals organized in a nacreous structure and are called nacre. The inner and middle layers are separated by a thin layer called a pellucid layer that is formed at impressions of an adductor and other muscles on a shell (Wada 1957). The prismatic and nacreous layers are produced by the outer epithelium of a mantle and are composed of calcium carbonate crystals and organic matrices made of proteins, polysaccharides, and lipids (Wilbur and Saleuddin 1983; Suzuki et al. 2007; Farre and Dauphin 2009).

In pearl production with cultured pearl oysters, a tissue fragment prepared from a pallial zone of a pearl oyster's mantle (donor) is implanted into the gonad of other pearl oysters (recipient). A small inorganic bead (pearl nucleus) made from the nacre of a freshwater mussel (family Quadrulidae) is also inserted and put in contact with the implanted mantle allograft. After the operation, the recipient starts to heal the wound. The recipient recognizes the mantle allograft and the bead as foreign materials. They are encapsulated by the hemocytes (granular and agranular hemocytes, Funakoshi 2000) of the recipient, and the hemocyte sheet is formed around the bead. The granular hemocytes phagocytose tissue debris, and the agranular hemocytes secrete extracellular matrices (ECMs, Suzuki et al. 1991; Suzuki and Funakoshi 1992). In the course of these wound healing processes, the outer epithelial cells of the mantle allograft become squamous and emigrate into the space between the bead and the surrounding hemocyte sheets. The outer epithelial cells finally form a follicle surrounding the bead that is called a pearl sac (Machii 1968). The outer epithelial cells of the pearl sac, now can be referred to as pearl-sac epithelial cells, start to form nacre, prismatic layers or organic matrices on the surface of the bead. Other tissue components of the mantle, such as muscles and the inner epithelia, remain for a while, but eventually disappear (Machii 1968). The processes of the pearl-sac formation resemble wound healing processes that occur after a mantle injury (Pauley and Heaton 1969; Armstrong et al. 1971; Sminia et al. 1973).

Processes of the shell and pearl formation summarized above have been elucidated mainly by histological observations, but recent biochemical and molecular biological studies on the organic matrix of the shell, especially organic matrix proteins (OMPs), have greatly increased the knowledge on the molecules involved in the shell and pearl formation (Zhang and Zhang 2006). In pearl oysters, primary structures of a variety of OMPs have been clarified. Based on the distribution or gene expression patterns, the pearl oyster OMPs are classified into three categories: OMPs present in the nacreous layer (Sudo et al. 1997; Samata et al. 1999; Kono et al. 2000; Miyashita et al. 2000; Bedouet et al. 2001; Zhang et al. 2006a; Ma et al. 2007; Gong et al. 2008c; Suzuki et al. 2009), in the prismatic layer (Sudo et al. 1997; Suzuki et al. 2004; Tsukamoto et al. 2004; Zhang et al. 2006b; Kong et al. 2009; Takagi and Miyashita 2010), and in both layers (Miyamoto et al. 1996; Kono et al. 2000; Zhang et al. 2003; Yano et al. 2006, 2007). Most of the OMPs are thought to undergo posttranslational modifications, and a detailed examination has been conducted for nacrein (Miyamoto et al. 1996), which is glycosylated with oligosaccharides that contain sulfite and sialic acid at its terminus (Takakura et al. 2008). There are some other OMPs, of which cDNA information has been released only in the public nucleotide database. Besides these protein components, the organic matrix of the pearl oyster contains a structural polysaccharide, chitin, as the other major component of the organic framework (Suzuki et al. 2007). Thus, the analyses on the components of the organic matrices of the pearl oyster shell have revealed the presence of an amazing variety of molecules that probably interact and coordinate for the formation and determination of the polymorphic shell structures (Matsushiro et al. 2003). Information on the OMPs is still increasing through transcriptome analysis of the mantle tissue (Liu et al. 2007; Jackson et al. 2010).

The function of the various components of the organic matrices in relation to the formation and determination of the polymorphic shell structures has been investigated by a combination of different approaches, such as in vitro calcium carbonate crystallization (Belcher et al. 1996; Falini et al. 1996; Zaremba et al. 1996) and in vivo monitoring of the entire process of shell formation by "flat pearl" (Fritz et al. 1994; Zaremba et al. 1996). Recent experiments conducted by Suzuki et al. (2009) have newly incorporated the RNA interference method to modify the synthesis of one of the matrix proteins, Pif, in the outer epithelial cells in vivo, and have clearly indicated the roles of Pif in nacre formation. These analyses have accumulated pieces of evidence, which demonstrate that the components of the organic matrices play crucial roles in the determination of the shell structures. However, considering the amazing elaboration of shell structures and the contribution of the outer epithelial cells to the shell formation, introduction of cell biological approaches is mandatory for further detailed analysis of the shell formation mechanisms. The outer epithelial cells play central roles in the shell formation. Inorganic ions for calcium carbonate crystallization are transported into the extrapallial space by these cells, and all the components of the shell matrices are synthesized and secreted in a precisely controlled manner from these cells. Environmental and physiological fluctuations are ultimately integrated in the outer epithelial cells and reflected in changes in the shell structures. As Zhang and Zhang (2006) have already pointed out, it is quite necessary to construct proper molluskan biomineralization-related cell models for further understanding of the formation and determination of the polymorphic shell structures.

The authors have been engaged in the development of tissue culture methods for mantle fragments and mantle outer epithelial cells of pearl oyster P. fucata (Machii and Wada 1989; Awaji and Suzuki 1998). During the course of the study, several basic methods for the tissue culture have been developed. Under in vitro conditions, cultured mantle fragments exhibited some phenomena that resemble secretion of organic matrices and calcium carbonate crystal formation by an intact mantle (Machii 1974). In addition, the importance of cell-to-cell interactions between the outer epithelial cells and hemocytes for pearl-sac formation was suggested through in vitro experiments using isolated outer epithelial cells and hemocytes (Awaji and Suzuki 1998). Although further improvements of the tissue culture methods are necessary for its application to the studies focusing on functions of the outer epithelial cells in the shell and pearl formation processes, the authors think that this is the appropriate time to summarize the progresses of in vitro studies on shell and pearl formation and reconsider the significance of the results for future studies.

In this article, the authors present first the results of their histological studies on pearl-sac formation processes of P. fucata and the relationships between morphological characteristics of pearl-sac epithelia and crystal structures. Secondly, tissue culture methods for pearl oyster mantle and some phenomena observed under in vitro culture conditions are described. Finally, an example of the applications of the tissue culture methods for pearl production is also presented.

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2. Outer epithelial cells of pearl oyster mantle in vivo

For pearl production, a fragment of the mantle tissue prepared from a donor pearl oyster is implanted into a recipient oyster, and the outer epithelial cells of the implanted mantle fragment start to form a pearl sac around the bead (Machii 1968). The behavior of the outer epithelial cells in the course of pearl-sac formation provides interesting and important information on the cell-to-cell interactions involved in the wound healing and cell proliferation in mollusks (Awaji and Suzuki 1998). After the completion of the pearl-sac formation, pearl-sac epithelial cells start to secrete materials for pearl formation. The pearl formation activities, however, vary greatly from a sac to another sac, and it is quite common to observe the difference in the secretive activities among pearl-sac epithelial cells even in a single pearl sac. Morphological characteristics of the pearl-sac epithelial cells show close relationships with the structures of adjacent pearl layers. In this chapter, the authors describe the histological characteristics and behaviors of the mantle outer epithelial cells in vivo.

2-1. Histological structure of the outer epithelium of the pearl oyster mantle

The soft body of a pearl oyster is enclosed and protected by a pair of shells from its lateral sides. The right and left mantles adhere to the inner surface of a shell of each side, holding the other part of the body in a mantle cavity between them. The interface between a shell and a mantle is called an extrapallial space (Wilbur and Saleuddin 1983). To observe the histological structures of a mantle, a young pearl oyster (50 mm in shell height) was anesthetized with 0.1% phenoxyethanol in seawater, and a mantle was fixed with Davidson's fixative. Tissue sections were stained with Mayer's hematoxylin and eosin or Giemsa stain.

A mantle of a pearl oyster has three folds, namely outer, middle, and inner folds, around the ventral edge of the marginal zone. The outer and middle folds adhere to each other to form a narrow slit called periostracal groove between them (Fig. 1A). Located at the bottom of the groove is a periostracum gland that synthesizes the periostracum (Figs. 1A, B). The periostracum, in general, is a thin proteinaceous sheet covering the outer surface of the molluskan shell (Saleuddin and Petit 1983). In pearl oysters, however, it is easily torn off, and it does not remain long on the shell surface. The proteinaceous components synthesized in the periostracum gland are cross-linked, modified by glycocalytic materials in the groove, and finally extruded from the groove (Fig. 1B). The membranous periostracum adheres to the shell edge, and it is thought to work to keep the extrapallial space in a closed condition favorable for shell formation. It is also thought to serve as the initial organic matrix for the deposition of calcium carbonate crystals (Saleuddin and Petit 1983).

The outer surface of a mantle, which faces a shell, is covered by a monolayer of epithelial cells (an outer epithelium) that play principal roles in shell formation (Fig. 1A). Three types of epithelial cells are recognized in the outer epithelium, among which the most abundant type is the outer epithelial cells (Tsujii 1960; Wada 1966). The outer epithelial cells in the marginal zone are columnar (Fig. 1A) and stained strongly with hematoxylin. Nevertheless, differences in the stainability of the epithelial cells are often observable as shown in Fig. 1C. Some of the outer epithelial cells with strong stainability to hematoxylin possess fine acidophilic (eosinophilic) granules in the cytoplasm (Fig. 1D). A nucleus of the outer epithelial cell is spherical or oval and possesses a prominent nucleolus. The cell height of the outer epithelial cells gradually decreases toward the pallial zone. In the pallial zone, there is a spot where the cell height changes sharply (Fig. 1E). The outer epithelial cells located dorsal to this zone sharply change their shape from columnar to cuboidal, decreasing their height to one third of the ventral cells. In addition, stainability of the cytoplasm to hematoxylin becomes weaker (Fig. 1E).


Fig. 1. Histological structures of the mantle of pearl oyster Pinctada fucata. A - transverse section of the marginal and the pallial zone of a mantle. A boundary between the marginal and pallial zones is indicated by a line. Outside of the mantle (o) is covered with a monolayer of epithelial cells stained in grayish blue with hematoxylin. An arrowhead indicates a periostracum gland. An arrow indicates the spot where the height of an outer epithelium changes sharply. m - marginal zone; p - pallial zone; of - outer fold; mf - middle fold; if - inner fold; pg - periostracal groove. V - ventral side; D - dorsal side; O - outside; I - inside. B - periostracal gland (arrowhead) and periostracal groove (pg). An arrow indicates periostracum. C - outer epithelial cells in the marginal zone. The outer epithelial cells that differ in the staining intensity with hematoxylin are observed. An arrowhead indicates outer epithelial cells strongly stained with hematoxylin. D - outer epithelial cells with fine acidophilic granules in the cytoplasm (arrowheads). E - a part of the pallial zone where the height of outer epithelial cells changes sharply. An arrowhead, corresponding to an arrow in A, indicates the spot where the cell height starts to change. Mayer's hematoxylin and eosin stain. Scale bars - 500 μm (A), 100 μm (B and C), 50 μm (D and E), respectively.

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There are two other types of cells in the outer epithelium: mucous cells and cells with large acidophilic granules (Fig. 2A). Mucous cells are characterized by a large vacuole in the cytoplasm and distribute over the entire outer epithelium. Although the cells with large acidophilic granules are relatively few in number, they can be found in and beneath the outer epithelium (Fig. 2A).


Fig. 2. A - presence of mucous cells (arrows) and cells with large acidophilic granules (arrowheads) in the outer epithelium. Cells with large acidophilic granules are also recognized beneath the outer epithelium. Giemsa stain. Scale bar - 50 μm. B - preparation of mantle fragments for pearl production. A ventral part of a mantle (v) is excised from a pearl oyster to prepare mantle fragments to be implanted. An inner side of the mantle is shown. After removal of the marginal zone (m), the remaining pallial zone (p) is cut into small pieces. Dark brown pigmentation is observable on the outer surface of the pallial zone (an arrow). B is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 134 pp., Fig. 5.9, © 1995, Shokabo Publishing.

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In pearl culture, small fragments of a mantle are prepared from a donor oyster to be implanted into recipient oysters. To prepare the mantle fragments, the ventral part of a mantle is excised from a pearl oyster, and the marginal zone is removed and discarded. The remaining pallial zone of the mantle is trimmed and cut into small pieces to utilize them for the implantation (Fig. 2B). Dark brown pigmentation is often observed over the outer surface of the pallial zone in healthy pearl oysters, typically as a thin blackish line parallel to the ventral edge (Fig. 2B). In pearl culture, this dark brown pigmentation is one of the conventional markers for the trimming of a pallial zone, and the mantle fragments for the implantation usually have this line in the middle of each of them. In histological observations of unstained sections of a pearl oyster mantle, dark brown pigments are sometimes observed in the cytoplasm of the outer epithelial cells at the spot where the cell height changes sharply. This implies that the pallial zone used to prepare the mantle fragments for pearl production corresponds to the area across the point where the epithelial cell height changes sharply.

2-2. Behavior of the outer epithelial cells in the course of pearl-sac formation

The pearl-sac epithelium is a monolayer of three types of cells as in an outer epithelium of mantle: outer epithelial cells, mucous cells, and cells with large acidophilic granules (Aoki 1966; Machii 1968). In the course of emigration and pearl-sac formation, the outer epithelial cells were thought to proliferate (Machii 1968), but the details of cell multiplication, such as distribution and frequency of mitotic cells, were not clear. The behavior of the two other cell types in the course of pearl-sac formation was also uncertain, although they have been reported to appear in the pearl-sac epithelium in later phases of its formation (Machii 1968). To clarify these issues, histological studies were conducted on the pearl-sac formation processes utilizing immunohistochemical methods for the detection of DNA synthesis in proliferating cells (Awaji and Suzuki 1995).

The experiment was conducted in June, and the seawater temperature ranged from 21–23°C during the experiment. Mature P. fucata, 70–80 mm in shell height, were kept in nets in the Gokasho Bay, Mie, Japan. A mantle fragment (2–3 mm square) was prepared from the pallial zone of a donor pearl oyster mantle, from which the inner epithelium had been removed, and was implanted into the recipient gonad through the transverse incision (8–10 mm) made into the gonad near the adductor muscle with a scalpel blade. An inorganic bead (7 mm in diameter), made from freshwater mussel shell, was also inserted and put in contact with the allograft. About 1 h after the operation, the recipient pearl oysters were returned to the nets.

Implantation of the inorganic bead (pearl nucleus) and mantle allograft caused rapid accumulation of hemocytes in the wound site. Two days after implantation (day 2), the bead and contacting mantle allograft had been completely encapsulated by layers of hemocytes (Fig. 3A). The allograft was still clearly discernible with flattening outer epithelium at the periphery of the fragment (Fig. 3B). Emigration of the epithelial cells from the allograft was still not observed. On day 4, the flattening of the outer epithelium proceeded further at the periphery of the allograft (Fig. 3C), and the squamous epithelial cells started to emigrate along the inside of the hemocyte capsule (Fig. 3D). The emigrating cells formed a continuous monolayer of epithelial cells covering the bead. It was difficult to distinguish between cell types because of the thinness of the pearl-sac epithelium. On day 8, the mantle allograft could not be distinguished from the surrounding tissue.


Fig. 3. Histological changes in mantle allografts and pearl-sac epithelia. A - hemocyte layers (arrowheads) encapsulating the implanted bead and surrounding testis (t) at 2 days after implantation (day 2). The bead was originally at b but removed for tissue sectioning. B - a mantle allograft (a) and surrounding testis (t) on day 2. Arrowheads indicate epithelial cells flattening at the periphery of the graft. C - a mantle allograft (a) on day 4. An arrowhead indicates flattening epithelial cells. D - a pearl-sac epithelium (arrowheads) emigrating from the allograft on day 4. E - a pearl-sac epithelium (arrowheads) on day 14. An arrow indicates acidophilic granules beneath the epithelium. F - mucous cells (m) and cells with acidophilic granules (arrows) in a pearl-sac epithelium (arrowheads) on day 21. b - the place in which the bead was located; o - ovary; t - testis. Mayer's hematoxylin and eosin stain. Scale bars - 160 μm (B, C), 80 μm (A, D), 40 μm (E, F), respectively. B is reprinted with permission from Fish. Sci., 61, Awaji and Suzuki, The pattern of cell proliferation during pearl sac formation in the pearl oyster, 747–751, Fig. 1a, © 1995, The Japanese Society of Fisheries Science.

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By day 14, the bead was completely covered with a monolayer of epithelial cells that had emigrated from the mantle allograft (Fig. 3E). Established pearl-sac epithelium consisted of outer epithelial cells and mucous cells—the former being dominant. Cells with large acidophilic granules could not be found in the pearl-sac epithelium, but clusters of acidophilic granules were found beneath the pearl-sac epithelium (Fig. 3E). On day 21, the number of mature mucous cells increased in most pearl sacs compared with earlier samples. The cells with large acidophilic granules could be found in the pearl-sac epithelium, and clusters of acidophilic granules were present under the pearl sac (Fig. 3F).

DNA synthesis is the most common parameter measured to estimate mitotic activity and cell proliferation. 5-Bromo-2'-deoxyuridine (BrdU) is an analog of thymidine and has been used to label the DNA of mitotically active cells. BrdU incorporated into nuclear DNA can be detected with an anti-BrdU antibody. In this study, BrdU was injected into pearl oysters at various phases of pearl-sac formation. Proliferation of the pearl-sac epithelial cells was examined in the course of pearl-sac formation by immunohistochemical staining for BrdU. Injection of BrdU was conducted every day, from 1 to 10 days, on 14, and 21 days after the operation. Each time, five oysters were randomly chosen from the operated animals, and 500 μl of 30 mM BrdU dissolved in marine molluskan balanced salt solution (MMBSS, Machii and Wada 1989, see table 3) was injected. At 24 h after BrdU injection, the part of the gonad in which the bead and the allograft were implanted was dissected out and fixed in 10% formaldehyde/seawater for 24 h at 4°C.

To determine the degree of mitotic activity of the outer epithelial cells in the pallial zone of a normal mantle tissue, BrdU was injected into unoperated control pearl oysters. In these animals, BrdU-labeled cells were rarely detected from the outer epithelium of the mantle, in contrast to the active labeling in the inner epithelium (Fig. 4).


Fig. 4. DNA synthesis in a mantle tissue from an unoperated pearl oyster. BrdU-labeled nuclei were visualized with anti-BrdU monoclonal antibody and FITC-conjugated second antibody. Note the absence of BrdU labeling in the outer epithelium (o) in contrast to active labeling (arrowheads) in the inner epithelium (i). Scale bar - 200 μm. Reprinted with permission from Fish. Sci., 61, Awaji and Suzuki, The pattern of cell proliferation during pearl-sac formation in the pearl oyster, 747–751, Fig. 2a, © 1995, The Japanese Society of Fisheries Science.

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When BrdU was injected on day 2, incorporation of BrdU was not observed at any part of the mantle allograft. When injected on day 4, however, BrdU-labeled nuclei were clearly detected in the flattening outer epithelial cells at the periphery of the allograft (Fig. 5A), in the epithelial cells emigrating from the allograft, and in the leading squamous cells (Fig. 5B). Some of the BrdU-labeled nuclei in the allograft were oval (3–4 μm in longer diameter) and sometimes had prominent nucleoli (Fig. 5A). In contrast, the incorporation of BrdU was very rare in outer epithelial cells in the central part of the allograft where the columnar structure of the epithelium was maintained (Fig. 5A). BrdU incorporation into the emigrating outer epithelial cells and squamous cells continued throughout the process of pearl-sac formation (Fig. 5C). On days 14 and 21, when the pearl sac had been established, the incorporation of BrdU was still observed in some of the outer epithelial cells in the pearl sac, although the frequency of incorporation was slightly lower compared with the earlier samples (Fig. 5D). No incorporation of BrdU was detected in mature mucous cells or the cells with large granules throughout the process of pearl-sac formation.


Fig. 5. DNA synthesis in allografts and pearl sacs. BrdU-labeled nuclei (colored brown) were visualized with anti-BrdU monoclonal antibody and horseradish peroxidase-conjugated second antibody. Black arrowheads indicate BrdU labeling in the epithelial cells. A - a mantle allograft (a) on day 4 (an adjacent section of Fig. 3C). Inset, enlargement of the epithelium shown by a right black arrowhead. Note active labeling of the nuclei. A white arrowhead indicates absence of BrdU labeling in the central part of the allograft. B - labeling of leading squamous epithelial cells on day 4. C, D - labeling of pearl-sac epithelial cells on day 14 (C) and day 21(D). b - the place in which the bead was located; o - ovary; t - testis. Scale bars - 160 μm (A), 80 μm (B, D), 40 μm (C), respectively.

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The process of pearl-sac formation observed in this study and the number of days required for its completion were almost the same as those reported previously (Machii 1968). The speed of pearl-sac formation has been reported to be strongly influenced by environmental water temperature (Aoki 1956; Machii and Nakahara 1957). This may be at least partly due to the effect of water temperature on the mitotic activity of pearl-sac epithelial cells.

Pearl-sac formation can be regarded as a phenomenon identical to the epithelial regeneration observed in cutaneous wound healing of mollusks (Pauley and Heaton 1969; Armstrong et al. 1971; Sminia et al. 1973; Suzuki et al. 1991). In this study, the outer epithelium of the mantle was shown to be mitotically inactive under normal conditions (Fig. 4). After the operation, however, the epithelial cells started to proliferate and regenerated the epithelium. These findings suggest that the outer epithelium is a stable tissue that begins active proliferation upon injury. In contrast, the inner epithelial cells proliferated frequently under normal conditions, indicating continual renewal of the tissue. Recently, however, Fang et al. (2008) have reported the presence of a proliferation hot spot in the outer epithelia of a central part of a mantle in P. fucata. The outer epithelial cells in the hot spot showed undifferentiated structure, and differentiation of the outer epithelial cells was suggested to proceed toward the marginal zone of a mantle. In the observations in this study on the DNA synthesis of an intact mantle tissue, a relatively ventral zone of a mantle was the target of the observations. There is a possibility that the proliferation hot spot was not in the area of the observations. However, in mantle allografts prepared from the pallial zone and implanted for pearl production, the outer epithelial cells started DNA synthesis in spite of the absence of the proliferation hot spot in the fragments. Therefore, the outer epithelial cells, even if they are located far from the proliferation hot spot, seem to maintain the ability to proliferate in response to injuries in a mantle.

Incorporation of BrdU into the mature mucous cells was not observed in the present study, which suggests low mitotic activity in these cells. Mitotic activity of immature mucous cells remained uncertain, as those cells could not be identified at the light microscopic level. Nevertheless, considering the fact that the number of mature mucous cells in the pearl-sac epithelium increased as the pearl-sac formation proceeded, the leading squamous cells probably included some immature mucous cells with mitotic activity that matured in the later phase of pearl-sac formation.

Incorporation of BrdU was not observed in the cells with large acidophilic granules. The origin of these cells in the pearl sac remains uncertain. However, on day 14, when the cells with large granules were still not present in the pearl sac, clusters of large acidophilic granules were found beneath the pearl-sac epithelium (Fig. 3E). After this phase, on day 21, the cells with large granules appeared in the pearl-sac epithelium (Fig. 3F). These observations suggest a relationship between the cells with large granules first observed on day 21 and the clusters of acidophilic granules beneath the pearl sac on day 14.

In the early phase of pearl-sac formation, incorporation of BrdU into the outer epithelial cells was very rare in the central part of the allograft where the columnar structure of the epithelium was maintained. It seems that the initiation of mitosis in the outer epithelial cells is closely related with their flattening and emigration. In humans, cutaneous wound healing processes have been shown to be a continually evolving network of interactions among cells, cytokines, and the extracellular matrices (ECMs), involving migration and proliferation of keratinocytes (Yamaguchi et al. 2005). In mollusks, most of the studies on wound healing have been histological, and the analysis of such dynamic interactions is limited. In pearl oyster, agranular hemocytes, which accumulated in the wound site, were shown to produce new ECMs on which new epithelium formed (Suzuki et al. 1991; Suzuki and Funakoshi 1992). The interactions among cells, cytokines, and ECMs are interesting issues to clarify, as these interactions appear to be involved in the process of wound healing and control of cell proliferation in mollusks.

2-3. Processes of pearl formation observed with the cover slip method

Histological observations on the processes of pearl-sac formation clarified changes in the morphology of the mantle outer epithelial cells together with the encapsulation of the implanted bead and the allograft by hemocytes. These observations, however, did not reveal initial events of pearl formation occurring on the surface of the bead. To realize the observations of the pearl formation processes in situ, a method of inserting a cover slip into an extrapallial space was developed (Wada 1961). With the application of this method for observation of pearl-sac formation processes, initial steps of calcium carbonate crystal formation and living cells committed for pearl-sac formation were observed (Machii 1968).

A small tissue fragment, 1–2 mm square, prepared from a pallial zone of a pearl oyster mantle was sandwiched between two cover slips cut into 5–8 mm square and transplanted into an incision (5–10 mm) made in an adductor muscle of a host oyster. The incision was made parallel to the bundles of muscles in the adductor muscle. After the transplantation, the cover slips with a mantle fragment were recovered periodically and observed under a phase contrast microscope. The experiments started in a period from May to August.

In 3 h after the implantation, numerous hemocytes appeared on the surface of the cover slips and around the edge of the mantle fragment. Two types of hemocytes were recognized: granular (GH) and agranular hemocytes (AGH). Of these two types, GH was a dominant cell type. GH was 10–22 μm in cell diameter with many granules in the cytoplasm and ac tively migrated on the surface of the cover slips. AGH were 5–15 μm in cell diameter when they were round but mostly triangular or star-shaped with extended pseudopodia. AGH slowly migrated on the surface of the cover slips or stayed still making connections with neighboring AGHs.

One to three days after the operation, the surfaces of the cover slips were covered with GH. At the peripheral part of the cover slips, clusters of hemocytes were observed. In the center of the cluster, GH and AGH were intermingled with each other, while AGH projecting long pseudopodia were observed at the periphery of the cluster. At the margin of the mantle grafts, epithelial-like cells migrated out from the explants forming an outgrowth zone (Figs. 6A, B). In some outermost parts of the outgrowth zone, scattering epithelial-like cells were observed. The epithelial-like cells were polygonal or round and with cell diameters of 10–20 μm. The outgrowth of the epithelial-like cells occurred 1 to 5 days after the operation, and continued thereafter.


Fig. 6. Observations of pearl formation with a cover slip method. A - outgrowth of epithelial-like cells (e) on day 1. m - mantle fragment. Phase contrast. B - emigrating epithelial-like cells in flattened appearance. Phase contrast. C and D - calcified crystals appeared on the glass surface on day 11 observed with a phase contrast (C) and a polarizing microscope (D). The crystals show birefringence under polarizing microscope. Arrowheads indicate identical crystals. Scale bars - 50 μm (A), 20 μm (B), 200 μm (C, D). B is reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Fig. 37, © 1968, Fisheries Research Agency, Japan.

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Four to seven days after the operation, all the surface of the cover slips was covered with epithelial-like cells, GH, and AGH. In some parts, epithelial cell sheets were formed on the glass surface, and dark brown organic precipitates started to appear on the glass surface at 7 days after the operation in an earliest case.

In 7 to 8 days after the operation and thereafter, epithelial-like cells of around 10 μm in cell diameter formed cell sheets covering all the surface of the cover slips. Under the epithelial cell sheets, layers of GH and AGH were observable. Calcified crystals showing birefringence under polarizing microscope started to appear on the glass surface at 11 days after the operation (Figs. 6C, D). When the cover slips were recovered from the pearl oysters in 2 or 4 months after the operation, a pair of the cover slips was completely covered and bound with nacre (Fig. 7).


Fig. 7. Cover slips completely covered with nacre. Operated in June and sampled after 2 months in August. Scale bar, 2 mm.

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Thus, the behavior of living outer epithelial cells and hemocytes during pearl-sac formation and the initial step of shell formation were observed by the cover slip method. The processes of the pearl-sac formation over the slides are illustrated in Fig. 8, which are consistent with those speculated from the histological observation of tissue sections of implanted mantle with bead using current commercial operation of pearl production as shown in Fig. 3. In addition, morphological appearance of calcified crystals on the cover slips, which was impossible to see in the methods of histological cross section of pearl sac with bead, could be observed. The shapes of the crystals were round or trefoil-like and resembled the crystals appearing in mantle fragment cultures; the details of which are described in Section 3.


Fig. 8. Processes of pearl-sac formation in the cover slip method. A - just after the implantation. B - the outer epithelial cells emigrate along the cover slips and proliferate. C - formation of a pearl sac. D - deposition of pearl layers around the cover slips. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Fig. 1, © 1968, Fisheries Research Agency, Japan.

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2-4. Relationships between morphological characteristics of pearl-sac epithelial cells and crystal structure of pearls

After the formation of the pearl sac, the epithelial cells of the pearl sac start to form pearl layers covering the bead. Appearance of pearls differs greatly by the crystal structures and organic components in the pearl layers. Most of the pearls from P. fucata can be classified into three types from their crystal structures on the surface: nacreous pearls, prismatic pearls, and organic (periostracal) pearls (Wada 1962). Surfaces of nacreous pearls and prismatic pearls are covered with nacreous and prismatic layers, respectively. Organic pearls are covered with dark brown secretions with or without calcium carbonate crystals. Concentric layers of different pearl structures are often observed around the bead (Wada 1962). Histological observations were conducted of decalcified pearls and pearl-sac epithelia to examine the morphological differences among the pearl-sac epithelial cells that produced the different morphological structures of pearls.

Pearl oysters P. fucata were implanted of an inorganic bead and a mantle allograft. After completion of the pearl-sac formation, the pearl sac and surrounding tissues were fixed with Davidson's fixative and decalcified by 3% trichloroacetic acid solution for about 4 months. The specimens were embedded in paraffin, and tissue sections were stained with hematoxylin and eosin or Azan for light microscopic observations.

Clear differences were observed in the morphology of the pearl-sac epithelial cells in relation to pearl structures as reported by other authors (Nakahara and Machii 1956; Aoki 1966). In samples forming a nacreous pearl, the organic matrices of decalcified nacre were recognized as concentric circles on the outermost surface of the implanted bead (Figs. 9A, D). Periodical emergence of relatively thick matrices was recognized in the matrix layers, implying presence of some rhythms in the nacre formation. The associating pearl-sac epithelial cells were in a squamous monolayer ranging 2–5 μm in thickness. The cytoplasm was slightly stained with hematoxylin, and cytoplasmic granules were scarce. Mucous cells were observed in the monolayer. In the connective tissues under the pearl sac, the cells with large acidophilic (eosinophilic) granules were observed.


Fig. 9. Decalcified pearls and adjacent pearl-sac epithelia. A–C - decalcified nacreous (A), prismatic (B), and organic (C) pearls and adjacent pearl-sac epithelia. Arrowheads in A - cells with large acidophilic granules; arrowheads in C - epithelial cells with acidophilic granules in the cytoplasm. n - nacreous layer; p - prismatic layer; o - organic layer; e - pearl-sac epithelium; m - mucous cell. D - a decalcified nacreous pearl and surrounding tissue. b - the space in which the bead was located. m - shell matrix of the implanted bead; o - organic matrix; p - prismatic layer; n - nacreous layer; e - pearl-sac epithelium; t - testis; d - digestive diverticula. Arrowhead - a transition layer between prismatic and nacreous layers. Scale bars - 20 μm (A), 150 μm (B), 250 μm (C) and 1 mm (D), respectively. D is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 161pp., Fig. 6.7, © 1995, Shokabo Publishing.

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In samples forming a prismatic pearl, thick organic matrices perpendicular to the surface of the bead were observed together with thinner matrices parallel to the surface (Fig. 9B). The pearl-sac epithelial cells were in a monolayer of 5–30 μm in thickness. The cytoplasm was slightly stained with hematoxylin. Some of the epithelial cells contained minute acidophilic granules in the cytoplasm. The mucous cells were found in the monolayer, and in some places, the mucous cells were observed in high density.

For the organic pearls, the matrices formed on the surface of the bead showed a variety of textures. Some were soft and membranous, but some were hard and carapace-like. Histological structures of the matrices varied accordingly. An example of soft membranous matrices was shown in Fig. 9C. In Azan stain, the fibrous matrices appeared as alternative layers of two differently colored substrates (Fig. 9C). One was stained blue with alcian blue, and the other was stained red with azocarmin G, which implied differences in their components. The associating pearl-sac epithelial cells were in a monolayer (Fig. 9C). The thickness of the epithelium was mostly 10–50 μm, but thicker (around 100 μm) or thinner (around 10 μm) cells were also observed. Some of the pearl-sac epithelial cells possessed many acidophilic granules in the cytoplasm. Cells resembling mucous cells with a vacuole and large acidophilic granules were observed in the pearl-sac epithelium. For hard carapace-like organic matrices, they were not stainable and showed dark brown color of their own.

Further histological observations of a decalcified pearl revealed that clear changes occur in pearl structures during the course of pearl formation. Figure 9D shows an example of the transitions of organic matrices from prismatic to nacreous. After the initial secretion of dark brown organic matrices (o in Fig. 9D), prismatic layers were formed around the bead which is stained red with eosin (p). After the continuous formation of prismatic layers, nacreous layers (n) were formed covering the whole surface of the prismatic layers. The interface between these structures was very clear. A thin matrix layer stained strongly in purple was observable between prismatic and nacreous layers, which indicated the presence of the transition layer reported by Wada (1957) and Dauphin et al. (2008). These observations imply sharp changes in the secretive activities of the pearl-sac epithelial cells.

Recently, primary molecular structures of a variety of shell matrix proteins are elucidated in P. fucata by cloning their cDNAs (Zhang and Zhang 2006). The expression of each matrix protein gene is restricted to epithelial cells in specific parts of a mantle tissue (Takeuchi and Endo 2005). Differences have been reported in expression patterns of these genes encoding several shell matrix proteins between mantle and pearl-sac epithelial cells, and among pearl-sac epithelial cells forming different types of pearls (Takeuchi and Endo 2005; Wang et al. 2009; Inoue et al. 2010, 2011a, 2011b). Daily oscillation in the gene expression is also reported (Miyazaki et al. 2008). However, there is still limited information on the relationships between morphology and gene expression of outer epithelial cells or pearl-sac epithelial cells. The results of the present histological observations of pearl-sac epithelial cells imply the presence of a specific gene expression pattern for each morphological characteristic and rapid transition from one state to others. Morphological observations combined with molecular biological analyses would contribute to clarify relationships between shell matrix proteins and pearl structures.

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3. Outer epithelial cells of pearl oyster mantle in in vitro organ culture

For further studies on the mechanisms of the pearl sac and pearl formation at cellular and molecular levels, it is desirable to use tissue culture methods because culture conditions can be controlled experimentally and changes in cultured mantles can be observed successively. Therefore, the authors have started working on developing tissue culture methods for the mantle of pearl oysters. This chapter presents the development of methods for the mantle fragment culture of P. fucata and the various morphological observations in culture conditions.

3-1. Development of methods for mantle fragment culture

Bevelander and Martin (1949), using P. radiata as material, conducted the first trial for mantle fragment culture of pearl oysters. They reported the deposition of conchiolin (shell matrices) and crystal aggregates typical of those found in normal and regenerating shells under tissue culture conditions. Unfortunately, details of their studies are not fully described in this short report. Following this early study, Machii and his colleagues have started to develop methods of mantle fragment culture for P. fucata (Machii 1974; Machii and Wada 1989). With the improvement of the culture methods, various phenomena related to shell formation have been observed in culture conditions. This section describes changes in the methods of mantle fragment culture for P. fucata chronologically, in order to provide information on developing culture conditions to study shell formation in vitro.

In early experiments (Machii 1968), a pallial zone of a mantle was excised from pearl oysters and sterilized by rinsing the strips in Cameron's saline for squids and clams (Solution A in Table 1) containing 80 U/ml penicillin and 80 μg/ml streptomycin. The mantle strips were cut into approximately 2 × 2 mm pieces, and the pieces were clotted onto the bottom of a culture dish with chicken plasma clot methods. The culture medium used for this study was a mixture of Cameron's saline, pearl oyster serum, adductor muscle extracts, and calf whole blood extracts as shown in Table 1. The culture temperature ranged from 18–28°C, and the cultures could be kept for 45 days in the best case.


Table 1. Tissue culture medium used for early studies on mantle fragment culture of pearl oyster Pinctada fucata. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Table 2, © 1968, Fisheries Research Agency, Japan.

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In this culture conditions, outgrowth of various types of cells occurred. At 4 h after the commencement of the culture, many granular and agranular hemocytes started active emigration from the mantle tissue fragments, and a small number of round epithelial-like cells, 20–25 μm in diameter, were observed around the fragment (Fig. 10A). Two days after the initiation of the culture (day 2), outgrowth of the epithelial-like cells to form cell sheets around the fragment together with granular hemocytes became apparent, but the attachment of the epithelial-like cells to the surface of the plasma clot was not firm. On days 4 and 6, cell densities of the epithelial-like cells in the outgrowth zones increased, and granular and agranular hemocytes were observed among the epithelial-like cells (Fig. 10B). At this time, spindle-shaped or elongated muscle cells started to emigrate from the fragments. On day 7 and afterward, continuous outgrowth of the above-mentioned types of cells was observable. During days 12–20, fibroblast-like cells, probably agranular hemocytes, formed reticular cell sheets at the peripheral part of the outgrowth zone. The mantle fragments were sub-cultured on around day 15, and in some cases, the outgrowth of the granular and agranular hemocytes and muscle cells continued. Secretion or deposition of the materials related to shell formation was not observed in these cultures.


Fig. 10. Fragment culture of pearl oyster mantle with the medium shown in Table 1. A - active outgrowth of granular and agranular hemocytes on day 1. m - mantle fragment. B - outgrowth of epithelial-like cells from the mantle fragment on day 4. Granular and agranular hemocytes are observable among epithelial-like cells. Phase contrast. Scale bars - 200 μm (A) and 100 μm (B), respectively. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Fig. 58, 60, © 1968, Fisheries Research Agency, Japan.

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After these early works, the culture medium was improved (Machii 1974) as shown in Table 2. Inorganic salt contents were based on Cameron's saline, and medium TC199 developed for mammalian cell culture was newly incorporated to the formula together with lactalbumin hydrolysate and calf serum. Small pieces of a sterilized mantle pallial zone were prepared as described above and clotted onto the bottom of culture vessels by plasma clot methods, and the newly formulated medium was added to the culture. The cultures were kept at 23°C in the gas phase of 10% CO2-90% air. Under these culture conditions, the outgrowth of the granular and agranular hemocytes, epithelial-like cells and muscle cells occurred in the same way as described above (Fig. 11). On day 12 and afterward, depositions of organic substances became recognizable on the mantle fragment or in the periphery of the tissue fragment in some cultures. The depositions were colored dark brown and first appeared as lines along grooves of the folds on the outer surface of the fragment. With the progress of the depositions, the mantle fragments were entirely colored dark brown (Fig. 12A). Histological observations of the mantle fragments in various degrees of the depositions revealed that the brown deposits start to appear in the sheets of epithelial cells outgrowing from the mantle fragments. Some fully colored fragments were entirely covered with dark brown membranes that could be stained with neither hematoxylin nor eosin (Fig. 12B).


Table 2. Tissue culture medium based on TC199 used for mantle fragment culture of pearl oyster Pinctada fucata. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 18, Machii, Organ culture of mantle tissue of the pearl oyster Pinctada fucata (Gould), 2111–2117, Table 1, © 1974, Fisheries Research Agency, Japan.

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Fig. 11. Fragment culture of pearl oyster mantle with the TC199-based medium shown in Table 2. Outgrowth of granular and agranular hemocytes, epithelial-like cells and muscle cells. Phase contrast. Scale bar - 100 μm. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 18, Machii, Organ culture of mantle tissue of the pearl oyster Pinctada fucata (Gould), 2111–2117, Fig. 2, © 1974, Fisheries Research Agency, Japan.

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Fig. 12. Deposition of dark brown secretions on the cultured mantle fragments. A - fragment culture of pearl oyster mantle in glass flasks. Some of the mantle fragments in the culture flasks are colored dark brown by the secretions (arrowheads). B - a cross section of the fully colored fragment. The fragment (m) is entirely covered with dark brown membrane-like structures (arrowhead). Delafield's hematoxylin and eosin stain. Scale bars - 10 mm (A) and 1 mm (B), respectively. A is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 179 pp., Fig. 6.15, © 1995, Shokabo Publishing.

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Following the deposition of the dark brown secretions on cultured mantle fragments, round or trefoil-like crystals started to be formed on the secretions. In some cultures, in addition, shell matrix-like organic frameworks were observed beneath the cell sheets. Details of these phenomena will be described in the next section.

Improvements of the culture media further proceeded (Machii and Wada 1989). The culture media employed up to this point were based on mixtures of components commonly used for vertebrate cell cultures. However, in vivo, molluskan mantle tissue is constantly supplied with circulating hemolymph and exerts its function under such conditions. Therefore, mantle fragments of pearl oysters maintained in vitro might show better performance in culture media with ingredients based on the hemolymph of their own. Based on this idea, Machii and his co-workers have analyzed salts, heavy metals, and free amino acids in the hemolymph of various mollusks, including P. fucata (Kawai et al. 1981). Then they formulated recipes for balanced salt solutions and tissue culture media based on the results of the analysis. The composition of the balanced salt solution for marine mollusks (MMBSS) and the medium for pearl oysters (Pf35) are indicated in Tables 3, 4, respectively.


Table 3. Balanced salt solution (MMBSS) and calcium/magnesium-free phosphate buffer (MMCMF) for marine mollusks. Used with permission of CRC Press, from Some marine invertebrates tissue culture, Machii and Wada, In: J Mitsuhashi (ed). Invertebrate Cell System Applications, Vol. II, 1989; permission conveyed through Copyright Clearance Center, Inc.

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Table 4. Tissue culture medium Pf35 for pearl oyster Pinctada fucata. Used with permission of CRC Press, from Some marine invertebrates tissue culture, Machii and Wada, In: J Mitsuhashi (ed). Invertebrate Cell System Applications, Vol. II, 1989; permission conveyed through Copyright Clearance Center, Inc.

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In mantle fragment cultures with Pf35, mantle fragments could be maintained for more than 4 months, and more active cell outgrowth was observed compared with previous results. Photographs in Figs. 13, 14 are records of a culture taken periodically during a period from day 7 to day 112. Cells from a mantle fragment outgrew actively (Figs. 13A–C), and many epithelial-like cells were recognized in the culture (Figs. 13D, E). The epithelial-like cells were polygonal in their shape, and possessed nuclei with a prominent nucleolus (Figs. 13F), which was a characteristic of mantle outer epithelial cells of P. fucata. Deposition of the dark brown secretions on the mantle fragments occurred in 2 weeks, which was followed by the crystal formation (Kawai et al. 1981; Machii and Wada 1989). The epithelial-like cells could be maintained for more than 3 months (Figs. 14A–C), and membranous structures with some minute precipitates could be observed under an outgrowth zone of the culture on day 112 (Fig. 14D). On the other hand, when the MMBSS medium was used, deposition of dark brown secretions could not be observed, although cell outgrowth from the mantle fragment also occurred on day 1 (Fig. 15A) and continued for several tens of days (Figs. 15B, C) (Kawai et al. 1981). These results indicate that the MMBSS composed of salts and glucose is not suitable enough for the deposition of dark brown secretions and the following crystal formation on the mantle fragments.


Fig. 13. Fragment culture of pearl oyster mantle with the hemolymph-based medium. A - active outgrowth of mantle cells from a fragment on day 49. B, C - active outgrowth of round epithelial-like cells on day 7 (B) and day 8 (C). D, E - outgrown epithelial-like cells attaching to the bottom of culture vessels on day 11 (D) and day 19 (E). F - round epithelial-like cells on day 26 possessing nuclei with a prominent nucleolus (arrowhead). m - mantle fragment. Scale bars - 1 mm (A), 400 μm (B, C, E), 200 μm (D) and 100 μm (F), respectively. D is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 161 pp., Fig. 6.14 right, © 1995, Shokabo Publishing. E is reprinted with permission from Protein, Nucleic acid and Enzyme, 34, Machii, Tissue culture of shell, 193–196, Fig. 3, © 1989, Kyoritsu Shuppan.

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Fig. 14. Fragment culture of pearl oyster mantle with the hemolymph-based medium. A–C - epithelial-like cells maintained in culture conditions for 37 days (A), 49 days (B) and 80 days (C) attaching to the bottom of culture vessels. D - membranous structures with some minute precipitates (arrowheads) observed under an outgrowth zone of the culture on day 112. The minute precipitates suggest initiation of crystal growth. m - mantle fragment. Scale bars - 400 μm (B, D) and 200 μm (A, C), respectively.

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Fig. 15. Fragment culture of pearl oyster mantle with MMBSS. A - active outgrowth of mantle cells on day 1. B, C - continued outgrowth of mantle cells on day 6 (B) and day 14 (C). m - mantle fragment. Scale bars - 1 mm (A) and 200 μm (B, C), respectively.

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Thus, the cells originating from the mantle fragments of P. fucata could be maintained for up to 4 months in culture conditions, and epithelial-like cells, resembling mantle outer epithelial cells, emigrated from the fragments attaching to the bottom of culture vessels and survived long. Interestingly, mantle fragments started to deposit dark brown secretions on their surface. Reproducibility of the dark brown secretion from mantle fragments has been confirmed in a different laboratory (Samata et al. 1994) by using Pf35 as a culture medium. To clarify components of the secretions, Yano and Machii (1975) analyzed amino acid compositions of the dark brown secretions formed on the cultured mantle fragments. The obtained amino acid composition, however, was dissimilar to those of any of the shell matrix proteins in the periostracum, prismatic layer, and nacre of P. fucata. Precise relationships of the dark brown secretion to shell formation processes is still not clear, but succeeding crystal formation on the mantle fragments implies that the secretion has some function related to shell formation.

3-2. Formation of crystals and shell matrix-like structures in mantle fragment cultures

As described in the previous section, round or trefoil-like crystals started to be formed on the dark brown secretions on the mantle fragments (Fig. 16A). The crystals have been characterized by the following: (1) distinct birefringence was demonstrated using a polarizing microscope (Fig. 16B); (2) X-ray absorption occurs as demonstrated by microradiogram preparations (Fig. 16C); and (3) laser microanalysis demonstrated strong calcium and magnesium absorption peaks (Machii and Wada 1989). The crystals were 100–150 μm in diameter, and their shape resembled prismatic crystals formed in vivo on the cover slips at the initiation of pearl formation as described in Subsection 2-3 (Figs. 6C, D). In some cultures, the crystals formed on the bottom of a culture vessel were observed (Figs. 17A, B).


Fig. 16. Round crystals formed on the dark brown secretions of a mantle fragment cultured for three months with the TC199-based medium. Arrows indicate identical crystals observed with three different methods. A - bright field microscopy of the crystals formed along grooves of the folds on the outer surface of the fragment. Arrowheads - dark brown precipitates deposited along the grooves. B - polarizing microscopy of the crystals. Distinct birefringence was demonstrated. C - X-ray absorption demonstrated by microradiogram preparations. Scale bars - 200 μm. B and C are reprinted with permission from Protein, Nucleic acid and Enzyme, 34, Machii, Tissue culture of shell, 193–196, Fig. 5.6, © 1989, Kyoritsu Shuppan.

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Fig. 17. Trefoil-like crystals formed on the bottom of a culture vessel. Mantle fragments were cultured for 1 month with the TC199-based medium. Phase contrast (A) and polarizing microscopy (B) of the identical crystals. Scale bars - 100 μm.

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Samata et al. (1994) analyzed the crystals formed on mantle fragments cultured with Pf35 medium by scanning electron microscopy (SEM) and energy dispersive X-ray microanalysis (EDS). Microcrystals of various shapes and sizes, containing large amounts of Ca and S, were detected in the specimens. The crystals were classified into three size classes. Crystals in the largest size group were larger than 50 μm in diameter and round in shape, which is consistent with the results presented in Machii and Wada (1989). Recently, Gong et al. (2008b) has reported in vitro calcium depositions observed in mantle fragment cultures of P. fucata. The depositions were formed on the mantle fragments and in the outgrowth areas. EDS analysis combined with SEM revealed that the deposits contained large amounts of calcium, carbon, and oxygen. They also detected nacrein, one of the shell matrix proteins (Miyamoto et al. 1996), secreted from the mantle fragment into the culture medium by western blotting. These results strongly imply that the cultured mantle fragments maintain a part of shell formation functions, and the materials secreted from the mantle fragments form the crystals.

Another interesting phenomenon observed in some mantle fragment cultures is the deposition of shell matrix-like organic structures beneath the cell sheet. Examples of the shell matrix-like structures formed on the bottom of culture vessels are shown in Figs. 18, 19. In these cultures, thin membranous structures were observable at peripheries of the cultured mantle fragments (Figs. 18A, B). In some parts, the structures appeared to overlap like scales (Figs. 18C, D). Minute spherical precipitates on these structures were observed in some cases (Fig. 14D). Polygonal frame-like structures resembling the shell-matrix of prismatic layers were also observed under the outgrowth zone of some cultures (Figs. 19A, B). Presence of a cell sheet or networks of epithelial-like cells over these structures was confirmed in some cases. As details of the microstructures and chemical compositions of these shell matrix-like structures are still not clarified, it is difficult to discuss these phenomena in detail. However, secretion of nacrein confirmed in the mantle fragment cultures (Gong et al. 2008b) implies simultaneous secretion of other shell matrix proteins, including water insoluble types, from cultured mantle fragments. Analysis of proteins and other components incorporated into the membranous structures is needed.


Fig. 18. Shell matrix-like membranous structures formed on the bottom of culture vessels. Mantle fragments were cultured for 1 (A, C, D) or 2 months (B) with Pf35 medium with slight modifications. Arrowheads indicate thin membranous structures formed on the bottom at peripheries of the mantle fragment. Overlapping of the structures is discernible in C and D. An arrow in A indicates dark brown secretions of the fragment. Scale bars - 200 μm (A–C) and 100 μm (D), respectively.

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Fig. 19. Polygonal frame-like structures observed under the outgrowth zone. Mantle fragments were cultured for 2 months with the TC199-based medium. A - frame-like structures (arrowheads) on the bottom of a culture vessel with covering cell sheets. B - frame-like structures after removal of the covering cells. Phase contrast. Scale bars - 200 μm (A) and 100 μm (B), respectively. B is reprinted with permission from Biomineralization (BIOM2001): Formation, Diversity, Evolution and Application. Machii, Pearl formation studies in vitro, 133–136, Fig. 4 left, © 2003, Tokai University Press.

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3-3. Proliferation of outer epithelial cells in mantle fragment culture

In the processes of pearl-sac formation, outer epithelial cells of a mantle allograft become squamous, migrate into the space between the bead and hemocyte sheets, and proliferate during the emigration. As mentioned in Subsection 2-2, the pearl-sac formation resembles epithelial regeneration in the cutaneous wound healing processes of mollusks. Cell-to-cell interactions via unknown bioactive molecules must be involved in these changes. If the early processes of pearl-sac formation can be reproduced in vitro, it will be easier to reveal the cell-to-cell interactions at cell and molecular levels. Therefore as a first step, the authors conducted histological observations of the cultured mantle fragments, including the detection of DNA synthesis by BrdU labeling methods.

A pallial zone of a mantle of P. fucata was excised and rinsed for sterilization as already described (Subsection 3-1). The tissue fragments in 3 × 3 mm were prepared and cultured in MMBSS supplemented with glucose (300 μg/ml) and kanamycin for 1 week (see Table 3). On day 6, BrdU was added to the culture medium, and the tissues were fixed after the labeling for 1 day. The tissue fragments were embedded in paraffin, sectioned, and stained for histological observations. BrdU incorporation into the nuclei of the outer epithelial cells was detected by immunohistochemical methods as described previously (Subsection 2-2).

On day 6, the outer epithelial cells at the edge of the mantle fragments were squamous and started to migrate along the edge of the fragments (Fig. 20A). Active BrdU incorporation was detected in these squamous epithelial cells (Figs. 20B, C). In the central part of the mantle fragments, in contrast, the outer epithelial cells remained in cuboidal shape, and DNA synthesis was not detected in these cells (Figs. 20A–C).


Fig. 20. DNA synthesis in the outer epithelium of a cultured mantle fragment. DNA synthesis was detected in the squamous outer epithelial cells emigrating from the edge (arrowheads) but not in the columnar outer epithelial cells in the central part of the fragment (arrows). A - Mayer's hematoxylin and eosin stain. B and C - immunohistochemical detection of BrdU-labeled nuclei in an adjacent section of A with anti-BrdU monoclonal antibody and FITC- conjugated second antibody. B - phase contrast microscopy. C - fluorescent microscopy. Scale bar - 200 μm.

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Results of the observations on outer epithelial cells in the mantle fragment cultures were analogous to the changes that occurred in outer epithelial cells during pearl-sac formation (Figs. 3B, C). The cultures were conducted using MMBSS supplemented with glucose as a medium. Addition of amino acids, vitamins, or other supplements, such as fetal calf serum, was not necessary. So flattening, migration, and proliferation of the outer epithelial cells seem to be triggered through cell-to-cell interactions inside the cultured mantle fragments. This implies the possibility to reproduce an early phase of the pearl-sac formation processes in vivo in tissue culture conditions in vitro. However, it is not easy to identify or restrict types of cells in the mantle organ culture. This makes the organ culture inadequate for the analysis of cell-to-cell interactions involved in the pearl-sac formation. As a next step, the authors tried to develop separation methods for outer epithelial cells; the results of which are presented in the next chapter.

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4. Outer epithelial cells of pearl oyster mantle in in vitro cell culture

In the processes of pearl-sac formation, the outer epithelial cells of a mantle allograft become squamous and proliferate (Machii 1968; Awaji and Suzuki 1995). Hemocytes that encapsulate the allograft and the bead secrete extracellular matrices in the cell sheets (Suzuki et al. 1991). After the pearl-sac formation, pearl-sac epithelial cells start to secrete shell matrices together with active transport of calcium and bicarbonate ions (Wilbur and Saleuddin 1983). How are these events stimulated to occur and controlled at a cellular level? To answer this interesting question, cell culture of the outer epithelial cells of pearl oyster mantle is an ideal tool to be employed. Following the development of the mantle-fragment culture methods, the authors have started their trials to isolate the outer epithelial cells from a pearl oyster mantle (Awaji 1991, 1997) and conducted some experiments to clarify cellular mechanisms of the pearl-sac formation processes (Awaji and Suzuki 1998). This chapter deals with the development of separation methods for the outer epithelial cells and the results of the experiments on proliferation of the outer epithelial cells in vitro, and on pearl production by injecting the cultured cells into pearl oysters with a bead.

4-1. Separation of outer epithelial cells of mantle

In order to collect sufficient amounts of outer epithelial cells of high purity and viability, an enzymatic digestion method to separate the outer epithelial cells in the form of cell sheets was developed (Awaji 1997). In addition, an enzyme suitable for single cell preparation from the isolated epithelial cell sheets was identified. These methods will be described in this section together with the histological structures of the isolated outer epithelial cells at light and electron microscopic levels.

Dissection and sterilization procedures of mantle fragments were illustrated in Fig. 21. A pearl oyster was opened by cutting the adductor muscle, and a visceral mass was carefully removed to leave the mantle adhered on the shell (Fig. 21A). The inner surface of the mantle was disinfected by serial treatment with 3% I2, 2% KI in 70% ethanol followed by 70% ethanol (Fig. 21B). The surface sterilized mantle was removed from the shell, and a pallial zone of the mantle was excised out (Fig. 21C). The obtained mantle strips were rinsed in MMBSS with antibiotics (200 μg/ml penicillin, 100 μg/ml Kanamycin) by gentle stirring with several changes of the medium (Fig. 21D). Most of the mucous secreted from the strips was detached from the strips by flushing the medium over the tissue with a pipette. As the inner epithelium was damaged by the surface sterilization, it was dispersed and removed during these washing procedures. The rinsed fragments were cut into 3–4 mm square pieces prior to enzymatic digestion. Dispase II (Sanko Junyaku, Tokyo) was dissolved to a concentration of 1000 U/ml in MMBSS containing 10% pearl oyster hemolymph, and the enzyme solution (5 ml) was usually used for digestion of a mantle fragment (approximately 0.4 g) for 5 h at 15°C with gentle shaking.


Fig. 21. Procedures of excision and sterilization of a pearl oyster mantle. A - an opened pearl oyster (left) and an oyster removed of a visceral mass (right). B - disinfection of the inner surface of the mantle. C - the pallial zone (hatched) to be excised from a mantle. a - adductor muscle; m - mantle; s - shell. D - washing and mucous removal of mantle strips in MMBSS. Reprinted with permission from Invertebrate Cell Culture, Awaji, Primary culture techniques for the outer epithelial cells of pearl oyster mantle, 239–244, Fig. 1, © 1997, Science Publishers.

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After the digestion with dispase, an outer side of the mantle fragment was easily recognized by the yellowish color of the epithelium over the fragment. Dark brown pigmentation was often observed, as the fragments were prepared from a pallial zone of a mantle (Figs. 22A, B). The outer epithelium confirmed was carefully and gently peeled off from the mantle fragment in MMBSS using fine forceps under a dissecting microscope. When the digestion was complete, the outer epithelium could be separated as perfect cell sheets (Fig. 22C). However, in a case of imperfect digestion, the cell sheets were smaller and aggregated as shown in Fig. 22D. Observations of the isolated epithelium with phase contrast microscopy showed that most of the cells in the sheet possess a nucleus with a prominent nucleolus that is typical of the outer epithelial cells of pearl oyster mantle (Fig. 22E). Dark brown pigmentation was also observable in some populations of the epithelial cells in the sheets (Fig. 22F). At higher magnifications, folds of the epithelium were observable (Fig. 23A). Histological observations of the cross section showed that the cell sheet is composed of the outer epithelial cells in a monolayer (Fig. 23B). Cell junctions between the outer epithelial cells were confirmed to be well maintained by observations with transmission electron microscopy (Fig. 23C). Further examinations of the isolated cell sheet with scanning electron microscopy indicated a monolayer structure of the sheet (Fig. 24A) and well-developed microvilli on the apical side of the outer epithelial cells (Fig. 24B). Interestingly, presence of an unknown type of cells with rod-like structures on the apical side was also shown (Fig. 24B).


Fig. 22. Separation of outer epithelium from a mantle fragment digested with dispase. A - a mantle fragment observed from the outside surface showing yellowish color of the epithelium. Dark brown pigmentation of a pallial zone (an arrow) is also observable. B - a lateral view of the digested fragment. The outer epithelium (arrowheads) is recognized by its coloration. Note difference in the color of the epithelium. C - outer epithelia peeled off from the mantle fragment in the form of cell sheets. The epithelial sheets have different colors. D - aggregates of small cell sheets having different colors. E - the separated outer epithelium observed with phase contrast microscopy. The epithelial cells possess a nucleus with a prominent nucleolus. F - bright field observation of the epithelium shown in E. Dark brown pigmentation is observable (arrowheads) in some populations of the epithelial cells. Scale bars - 1 mm (A-D), 100 μm (E, F). C is reprinted with permission from Invertebrate Cell Culture, Awaji, Primary culture techniques for the outer epithelial cells of pearl oyster mantle, 239–244, Fig. 2B, © 1997, Science Publishers.

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Fig. 23. Histological structures of the separated outer epithelia. A - binocular observations showing the folds of the epithelium. B - cross section of the epithelium showing a monolayer structure. a - apical surface with microvilli; b - basal surface with some connective tissue cells. C - transmission electron microscopy of the epithelium. Intercellular attachment between the epithelial cells (arrowheads) remained intact after the dispase digestion. m - mitochondria; n - nucleus; rER - rough endoplasmic reticulum. Scale bars - 500 μm (A), 50 μm (B) and 3 μm, respectively. B is reprinted with permission from Invertebrate Cell Culture, Awaji, Primary culture techniques for the outer epithelial cells of pearl oyster mantle, 239–244, Fig. 2C, © 1997, Science Publishers.

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Fig. 24. Scanning electron microscopy of the separated outer epithelium. A - a piece of separated epithelium showing a monolayer structure. On the apical side (a), the top of each epithelial cell is discernible as a cupola. b - basal side. B - well-developed microvilli on the apical surface of the separated epithelium. Arrowheads - cells with rod-like structures.

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The separated epithelia were often surrounded by a mucous-like substance, which interfered with the collection of the epithelia by centrifugation. Therefore, the obtained epithelia were washed in MMBSS containing 1 mg/ml collagenase (from Clostridium histolyticum, Wako, Osaka), 4 mg/ml DNAse I (Sigma DN-25), and 0.4 mg/ml testicular hyaluronidase (Sigma H3506) by gentle stirring. These additional washing steps broke the epithelial sheets into cell clumps that could be collected by centrifugation at 130 g for 5 min.

The recipe of the dispase solution and conditions of the digestion were recently modified for better performance. At present, 4 U/ml dispase (from Bacillus polymyxa, Invitrogen) and 0.5 mg/ml collagenase (from Clostridium histolyticum, Wako) dissolved in MMBSS buffered with 10 mM HEPES (pH 7.3) are used for the tissue digestion at 25°C for 5 h with gentle shaking. The additional washing before collection of the cell clumps by centrifugation is conducted in MMBSS buffered with 10 mM HEPES (pH 7.3) containing 0.4 mg/ml hyaluronidase (from ovine testes, Wako). The use of DNAse I has been omitted from the washing procedures, as it might interfere with later molecular biological analyses.

Histological observations described above indicate that the outer epithelial cells can be separated from a pearl oyster mantle in the form of cell sheets by dispase digestion. The separated outer epithelia were in a monolayer with little contamination by connective tissue cells or hemocytes. Isolation of the outer epithelial cells in cell sheets is probably beneficial for the studies on shell formation and pearl-sac formation, as the isolated epithelial cells maintain their polarity and cell junctions between the cells. However, there are also some disadvantages in the dispase digestion method. Major problem lies in the fact that the epithelial cells are collected finally in the form of cell clumps. For quantitative studies using cell culture, seeding of target cells at a decided concentration is always required. Cell adhesion to culture plates is greatly influenced by the initial attachment of the cells to the bottoms. In these aspects, outer epithelial cells dissociated into single cells are more preferable. Therefore, for better performance of the outer epithelial cell cultures, proteases suitable for dissociation of the epithelial cell clumps were examined. Among various proteases tested, pronase (from Streptomyces griseus, Calbiochem) was most effective, and digestion of the epithelial clumps at 10 mg/ml in MMBSS buffered with 10 mM HEPES (pH 7.3) for 1 h at 25°C was suitable for the dissociation (Fig. 25).


Fig. 25. Dissociation of the outer epithelial cell clumps by pronase at various concentrations. Digestion of the epithelial clumps at 10 mg/ml was suitable for the dissociation. A - 1 mg/ml; B - 5 mg/ml; C - 10 mg/ml. Scale bars - 100 μm.

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For purification of the outer epithelial cells from a pearl oyster mantle, density gradient centrifugation of the mantle cells is an alternative. Recently, Gong et al. (2008a) reported cell culture of mantle outer epithelial cells of P. fucata. They purified the outer epithelial cells from the cells migrated out in mantle explant cultures. Identification of epithelial cells was conducted with a monoclonal antibody to pancytokeratin, and an anti-nacrein antibody detected the secretion of a shell matrix protein (nacrein, Miyamoto et al. 1996) into the culture medium. These results indicate that the outer epithelial cells are collectable by density gradient centrifugation. In our studies, purity of the outer epithelial cells was confirmed by detailed histological examinations of the isolated cells. For in vitro quantitative studies, however, it is better to incorporate specific molecular markers for the outer epithelial cells in addition to morphological characteristics and functional markers related to shell formation as shown in Gong et al. (2008a) for instance.

Recently, cDNA structures of various shell matrix proteins have been elucidated in P. fucata. Expression of these shell matrix protein genes will be one of the functional markers for the outer epithelial cells. Extraction of total RNA from the outer epithelia separated by the dispase method (without DNAse I treatment) and the following synthesis of cDNA for polymerase chain reactions (PCR) could be performed without any problems. PCR with specific primers for shell matrix proteins of P. fucata (nacrein, Miyamoto et al. 1996; MSI60, MSI31, Sudo et al. 1997) generated corresponding DNA fragments (data not shown). Short-time incubation of the separated outer epithelial cells will be an interesting tool for screening of bioactive substances affecting gene expression of shell matrix proteins.

4-2. Proliferation of outer epithelial cells of mantle in co-culture with hemocytes

Histological studies have revealed that outer epithelial cells of the mantle allograft migrate attaching their basal side to the hemocyte sheet and proliferate. Based on these observations, the authors hypothesized that the morphological changes and DNA synthesis of the outer epithelial cells would be stimulated through interaction with hemocytes infiltrating the wound site. To ascertain this hypothesis, in vitro experiments were conducted using the outer epithelial cells isolated from the pearl oyster mantle (Awaji and Suzuki 1998).

Epithelial cell clumps were prepared from pearl oysters as described in the previous section, and the clumps were suspended in MMBSS. Cell density was determined by staining cell nuclei with 0.1% (wt/vol) crystal violet in 0.1 M citric acid (Sanford et al. 1951).

To prepare hemocytes for the experiment, blood was aseptically collected from adductor muscle of pearl oysters using sterile plastic syringes and cooled on ice in a sterile polypropylene tube. Collected blood was dispensed into a 24-well culture plate at 2.5 ml for each well. The plate was left still at room temperature for 4 h, during which hemocytes (1–2 × 106 cells/ml) sank to the bottom and formed a semi-confluent cell sheet with some aggregates. After the formation of the hemocyte sheet, hemolymph was removed from the wells. The mantle epithelial clumps suspended in MMBSS (1–2 × 106 cells/ml) were immediately added to the wells at 0.5 ml/well and cultured at 20°C for 7 days.

Epithelial clumps adhered to the hemocyte layers within 24 h (Fig. 26A). At the same time, the necrotic epithelial cells that were liberated from the clumps became spherical in shape and floated over the hemocyte layers. Some of the hemocytes also became spherical and detached from the hemocyte layers. These floating cells covered the entire bottom of the well. On day 1, some of the epithelial clumps started to change shape from cuboidal to squamous (Fig. 26B). The peripheral border of the clumps became less discernible under a phase contrast microscope, and migration of epithelial cells could also be detected at this stage (Fig. 26C). On day 7, the presence of small epithelial cell sheets outgrowing from the epithelial clumps could be detected (Fig. 30).


Fig. 26. The epithelial clumps cultured on a hemocyte sheet. A - 1 h after the inoculation. The epithelial clumps (arrows) have started to adhere to the hemocyte sheet (h). B and C - 1 day after the inoculation. In B, the floating cells liberated from the epithelial clumps and hemocyte sheets are covering the bottom of the well. Arrow - an epithelial clump changing its shape. In C, the epithelial cells have started to migrate (arrows) from the clump (c). Scale bars - 200 μm (A) and 100 μm (B, C), respectively. With kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 2A–C.

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Electron microscopy showed that the epithelial cell sheets formed on the hemocyte layers were in a monolayer (Fig. 27). They adhered to the hemocyte layers at the basal side and possessed microvilli on the apical side (Figs. 27, 28A). Cell-to-cell junctions (e.g., desmosomes and septate junctions) were well maintained between the migrated epithelial cells (Fig. 28B). At the peripheral part of the epithelial sheet, the epithelial cells became extremely flattened and acellular spaces often occurred between hemocyte layers and the epithelial sheet (Fig. 27B). Even in these cases, cell-to-cell junctions were maintained, and microvilli were observed at the apical side of the epithelial cells (Fig. 28C).


Fig. 27. Monolayer epithelial cell sheets formed on the hemocyte layers. The outer epithelial cells (e) adhered to the hemocyte layers (h) at the basal side, and possessed microvilli (arrowheads) on the apical side - A, a relatively thick monolayer of the epithelial cells at the central part of the cell sheet. B - the peripheral part of the epithelial cell sheet. The epithelial cells became flattened, and acellular spaces often occurred between hemocyte layers and the epithelial sheet. C - an extremely flattened epithelial cells at the margin of the cell sheet. p - bottom of the plastic well. Scale bars - 5 μm.

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Fig. 28. Ultrastructure of the epithelial cell sheet. A - central part of the epithelial cell sheet. B - cell-to-cell junction observed in the epithelial cell sheet. C - cell-to-cell junction of the squamous epithelial cells. e - the epithelial cell sheet; h - hemocyte layer; m - microvilli; s - septate junction; d - desmosome. Scale bars - 1 μm (A) and 0.5 μm (B, C), respectively. B and C are reprinted with kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 3B, D.

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The effects of the hemocyte layers on the adhesion of the mantle epithelial cells were confirmed by the enumeration of the adhering cells (Table 5). As it is difficult to distinguish the adhering epithelial cells from hemocytes, the number of the adhering epithelial cells was estimated from the difference between the results of two wells: the experimental well with epithelial cells on the hemocyte layer and the control well with only hemocyte layer (see B and C of Table 5). The presence of the living hemocyte layers clearly enhanced the adherence of the epithelial cells compared with the absence of hemocyte layers (compare A and B in Table 5, Fig. 29A). The hemocyte sheet fixed with 10% formaldehyde/seawater could also enhance the adhesion of the epithelial cells (compare A and C in Table 5), but the effects were weaker than the living cells (compare B and C in Table 5). The monolayer formation did not occur on the fixed hemocyte layers (Fig. 29B).


Table 5. Effects of the hemocyte layers on the adhesion of the mantle epithelial cells. With kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Table 1.

aThe mantle epithelial cells and hemocytes were inoculated at 1.0 × 106 and 6.1 × 106 cells/well, respectively. In A, the epithelial cells were inoculated to the wells without the hemocytes. In B and C, the hemocyte layers were prepared in the wells. Hemocytes were fixed with formaldehyde in the case of C. The epithelial cells were inoculated onto the hemocyte layers. The wells without the epithelial cells served as the controls. b×106 cells/well. cMean ± standard error; n = 3. dThe enumeration with crystal violet/citric acid solution required liberation of the stained cell nuclei, but only a small portion of the fixed hemocytes liberated the cell nuclei.

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Fig. 29. A - the epithelial cell clumps inoculated into a plastic well without hemocytes. B - the epithelial clumps cultured on the fixed hemocyte sheet on day 7. The epithelial cell clumps (e) adhered to the fixed hemocytes (h) but did not form a monolayer sheet. Spherical cells were liberated from the clumps. Scale bars - 100 μm. B is reprinted with kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 2D.

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To detect DNA synthesis of the epithelial cells, a BrdU labeling test was carried out on days 6–7. Cells with nuclei immunoreactive to anti-BrdU antibody were detected from the epithelial cell sheets outgrowing from the epithelial clumps onto the hemocyte layers (Fig. 30). Some weakly immunoreactive nuclei and non-immunoreactive nuclei were also observed in the same epithelial cell sheet. The frequency of the immunoreactive cells was variable by the cell sheets (Figs. 30A–C), and in a few cases, the sheets did not include the positive cells (Fig. 30D). Immunoreactive nuclei in the clumps were difficult to recognize because of the high background staining of the clumps.


Fig. 30. Detection of DNA synthesis in epithelial cell sheet. The cultures on day 6 were labeled with BrdU for 24 h and immunohistochemically stained with anti-BrdU antibody to visualize DNA synthesis in the cells. A part of the immunoreactive nuclei is indicated by black arrowheads. White arrowheads indicate nuclei without positive reactions. A–C - the epithelial cell sheet with different sizes and labeling frequency. D - the epithelial cell sheet without positive reactions. Scale bars - 100 μm. A and B are reprinted with kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 4.

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Thus, the mantle outer epithelial cells cultured on the hemocyte layers adhered and migrated on the hemocytes, changing shape from cuboidal to squamous. They eventually formed monolayer sheets. Cell polarity and cell-to-cell junctions were well maintained in the newly formed epithelial sheets. These morphological features of the migrating epithelium resemble those described for the outer epithelial cells in pearl-sac formation (Machii 1968; Awaji and Suzuki 1995). Effects of bioactive substances exogenously added to the medium on the morphological changes can be neglected, as BSS supplemented only with glucose was utilized for the culture. In addition, commencement of DNA synthesis, which is a prerequisite for cell multiplication, was also confirmed in the monolayer epithelial cells. The obtained results support the hypothesis that the interaction between outer epithelial cells and hemocytes is essential for the proper progress of pearl-sac formation and molluskan cutaneous wound healing.

The epithelium-hemocyte interaction observed in the co-culture system can be divided into three consecutive processes: epithelial adhesion to hemocytes, epithelial flattening and migration, and DNA synthesis. As for the epithelial adhesion, the importance of the hemocyte sheet was shown in the epithelial adhesion assay. Inoculated epithelial clumps could adhere to both living and fixed hemocyte layers, but the adhesion was firmer in the former case. Moreover, epithelial flattening and migration occurred only on the living hemocyte layers. The authors interpret the present data as indicating that the hemocyte sheet supplies matrices for the epithelial adhesion and migration. The hemocyte sheet, which is formed at in vivo wound sites, probably has the same function (Suzuki et al. 1991). The effects of fixation may imply that the matrices were susceptible to formalin. Alternatively, stable adhesion may be attained through successive interactions (e.g., cell movement and new matrix production) between the living epithelial cells and the hemocytes.

It is well known that coating a tissue culture dish with extracellular matrices (ECMs), such as collagen and fibronectin, improves the adherence of epithelial cells to the dish. As for molluskan ECMs, collagens have been purified from various tissues (Kimura et al. 1981; DeVore et al. 1984; Mizuta et al. 1994; Yoneda et al. 1999). Suzuki and Funakoshi (1992) reported the presence of collagen and gelatin-binding protein (fibronectin-like molecule) in the hemocyte sheets at wound sites. They also reported synthesis of collagen in hemocytes cultured in vitro (Suzuki et al. 1991). Effects of molluskan ECMs on the adhesion and migration of epithelial cells are interesting issues to be addressed, but at present, ECMs that enhance the adhesion of outer epithelial cells have not been elucidated. The reason for acellular spaces observed between the amebocyte layer and the peripheral epithelial sheet is not clear. It might be due to an artifact of the histological procedures, the attachment being so weak that the two cell layers became separated because of shrinkage. There is also a possibility that the outer epithelial cell sheets maintain functions for the transport of ions such as Ca2+ or HCO3 (Wilbur and Saleuddin 1983).

In the mammalian cutaneous wound healing processes, many types of cells communicate with each other through cytokines. Bioactive peptides, such as transforming growth factor-β, bone morphogenetic protein, basic fibroblast growth factor, and keratinocyte growth factor, are known to mediate mesenchymal-epithelial interactions in the skin, which leads to epithelial regeneration (Yamaguchi et al. 2005). In mollusks, the presence of some growth factor peptides has been reported (Hermann et al. 2000; Lelong et al. 2000; Akalal et al. 2003). Further clarification of molluskan growth factors and ECMs and their functional analyses will contribute to the development of tissue culture methods for molluskan cells.

4-3. Pearl production with cultured outer epithelial cells of mantle

The primary purpose of mantle tissue culture of pearl oysters is to study shell formation processes in simplified conditions and to elucidate mechanisms controlling the processes. In addition to the application for these basic studies, cultured outer epithelial cells might also be valuable for pearl culture industries. In the current methods of pearl production, a mantle fragment is transplanted into a host oyster together with an inorganic bead. The outer epithelial cells form a pearl sac, but the other parts of the mantle allograft degenerate and eventually disappear, probably through phagocytosis by hemocytes of recipient pearl oysters (Machii 1968). This inflammatory reaction to the transplanted tissue sometimes causes heavy accumulation of hemocytes in the wound sites, which often leads to the undesirable formation of organic layers on the pearl surface. Therefore, it might be preferable to remove the unnecessary part of the tissue from the mantle fragment. One possible alternative improvement of the current surgical procedure would be the implantation of only mantle epithelial cells instead of a mantle fragment into a recipient pearl oyster.

Machii and his colleagues conducted some experiments related to this idea (Machii 2007). Mantle fragments of P. fucata were cultured by previously described methods for 1 day (Figs. 31A), and cells that outgrew from the fragments were harvested by digestion of cell sheets with pronase. The cells were washed in MMBSS, and the obtained cell suspensions containing epithelial-like cells, granular and agranular hemocytes, and muscle cells (Figs. 31B) were injected into the host oyster implanted with an inorganic bead in advance. Among the 38 pearl oysters used for the study, 13 animals held the implanted bead in their soft body after 1 year. One of the 13 beads retrieved from the oysters was completely covered with nacreous layers, but the others were without pearl layers of any kind (Figs. 31C).


Fig. 31. Trial of pearl production with cultured mantle cells. A - cells outgrowing from the mantle fragment on day 1. B - cell suspension used for the injection. Cells outgrown from the mantle fragments (shown in A) were harvested by digestion of cell sheets with pronase. C - one of the 13 beads retrieved from the oysters was completely covered with nacreous layers (arrowhead), but the others were without pearl layers of any kind. Scale bars - 100 μm.

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These results indicate that the cultured outer epithelial cells retained the ability to form a pearl sac around the bead and secrete pearl layers on the bead. Although the percentage of pearl formation in this trial was very low, it might be possible to improve the production rate by using purified outer epithelial cells at higher cell densities. As the cells used for this experiment were cultured only for 1 day, it is not clear if shell formation functions of outer epithelial cells are maintained in longer cultures. Improvements of culture conditions will be necessary for practical application of the cultured outer epithelial cells to pearl production. However, various phenomena hitherto reported in the mantle fragment or mantle epithelial cell cultures imply that application of the cultured outer epithelial cells for pearl production is promising.

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5. Future prospects

It is clear that the outer epithelial cells play central roles in the shell and pearl formation. However, functions of the outer epithelial cells, such as ion transport, shell matrix synthesis and secretion, and the control mechanisms of these processes, are still not well understood. Without clarifying these activities of the outer epithelial cells, the understandings on formation and determination of the shell and pearl structures will remain inadequate. To address these issues, development of tissue culture systems for molluskan biomineralization studies is required.

Since the first trial of mantle tissue culture of pearl oysters by Bevelander and Martin (1949), methods for the mantle tissue culture have advanced step-by-step in spite of many difficulties that had to be overcome. A major problem has been the contamination with microorganisms. Especially, contamination with marine zoosporic fungi of unidentified types (Figs. 32) was a serious problem because of their active growth in cultures of molluskan and other marine invertebrate tissues (Machii et al. 1988). However, owing to the need for a novel model system for the elucidation of molluskan shell and pearl formation mechanisms, mantle tissue culture is now becoming one of the leading study areas in molluskan tissue culture (Suja and Dharmaraj 2005; Suja et al. 2007; Gong et al. 2008a, b).


Fig. 32. Zoosporic fungi appearing in a cell culture from veliger larvae of P. fucata. Cell clusters resembling animal cells are all fungus cells. The clusters attach to the bottom firmly by many pseudopodia-like extensions observable around the cell clusters (arrowheads). Scale bar - 100 μm. Reprinted with permission from Commun. Appl. Cell Biol., 15, Machii, In vitro studies of pearl formation, 1–10, Fig. 7, © 1998, Japan Academy of Applied Cell Biology.

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Cell culture of outer epithelial cells is more advantageous than mantle fragment culture for molecular and cell biological analyses of shell formation because it is quantitative, suitable to control cell types in the cultures, and compatible with the latest molecular and cell biological methods. For instance, Gong et al. (2008a) have developed an enzyme-linked immunosorbent assay system for one of the organic matrix proteins, nacrein, and observed the effects of Ca2+ concentration of the culture media on nacrein secretion in in vitro cell culture. By the application of various novel methods established in vertebrate or insect cell cultures, functions of outer epithelial cells in shell and pearl formation will be elucidated more in detail.

A shell-matrix protein gene shows a gene-specific positional pattern of expression in an outer epithelium (Takeuchi and Endo 2005). Control mechanisms for such a gene expression are currently obscure, but one possibility is that the expression of a matrix protein gene in an outer epithelial cell is influenced by the crystal structure of the shell adjacent to the epithelial cell. There is also a possibility that cell-to-cell interactions between the cells in the epithelium and the surrounding tissue affect the gene expression. Recently, Suzuki et al. (2009) have adopted RNAi technology to clarify the function of a nacre-specific matrix protein and have clearly indicated that the knockdown of the gene of acidic matrix protein Pif in vivo affects the crystal structures of nacre. Control mechanisms of shell-matrix protein gene expression will be an important area in future studies on pearl formation. In vitro culture of the outer epithelial cells will provide various possibilities to examine and manipulate the expression of shell-matrix protein genes in the outer epithelial cells.

Endocrinological control of shell formation is a well-known phenomenon observed mainly in freshwater gastropods (Geraerts 1976; Dogterom et al. 1979; Kunigelis and Saleuddin 1985). In freshwater snail Lymnaea stagnalis, a specific type of neurons (light green cells in cerebral ganglia, LGCs) controls body and shell growth of the snail (Geraerts 1976). As molluskan insulin-related peptides (MIPs) are produced in LGCs and released into the hemolymph, MIPs are candidate hormones for the control of body and shell growth of the snail (Smit et al. 1988). Bioactive peptides controlling body and shell growth are not well clarified in bivalves, but several factors that might be involved in the control of shell formation have been reported (Cudennec et al. 2006), including cDNA structures of insulin-related peptide in Pacific oyster Crassostrea gigas (Hamano et al. 2005) and scallop Mizuhopecten yessoensis (GenBank AB125891). In vitro culture of the outer epithelial cells will be a model system suitable for examining the effects of candidate peptides on cell function and clarifying endocrinological control mechanisms of shell formation.

Although the separation of outer epithelial cells has become possible (Awaji 1997; Gong et al. 2008a), further technical developments for the in vitro culture of outer epithelial cells are needed. Especially, studies on suitable culture substrates (extracellular matrices) and basal media are essential. These factors will not only affect the survival and growth of the cultured cells but also influence the functions related to shell formation. Three-dimensional culture of outer epithelial cells is also a method that has to be examined. Animal cell culture is usually conducted as a monolayer culture of the target cells. However, some types of differentiated cells require a three-dimensional environment, such as collagen gel culture, for the maintenance of their functions. In addition, shell and pearl formations proceed in a closed environment, such as an extrapallial space or inside of a pearl sac. Therefore, outer epithelial cells in a closed follicular structure might be a better model system for shell formation studies than the epithelial cells in a monolayer sheet.

Cell culture of outer epithelial cells is not only for basic studies on the shell and pearl formation processes but also for practical applications in the commercial production of pearl. Recently, Suja and Dharmaraj (2005) and Suja et al. (2007) have reported mantle tissue culture of abalone Haliotis varia, and they have applied the developed methods for the production of abalone pearls in tissue culture conditions (Dharmaraj and Suja 2006). This patent makes us realize that the cultured outer epithelial cells of P. fucata may become practically applicable for pearl culture in future. A possible style of the practice has been shown in the last section of this article. There are, however, many issues to be solved before the practical application is implemented. For example, it is difficult at present to make outer epithelial cells of pearl oyster mantle continue to proliferate in culture conditions, and the maintenance of shell formation functions in in vitro conditions for a long period is also uncertain. Recently, information on molluskan growth factors and growth factor receptors is accumulating (Hermann et al. 2000; Lelong et al. 2000; Akalal et al. 2003; Zhou et al. 2010). It will be important to study the effects of various growth factors on the multiplication of outer epithelial cells in culture conditions and the effects of various media supplements on cell growth and function. Although successful application of cultured outer epithelial cells for pearl culture still requires the accumulation of various types of knowledge on the functions of outer epithelial cells, it will revolutionize the methods of pearl culture. For example, selection of the cells for implantation with various favorable pearl formation abilities, genetic improvement of the cells by molecular biological methods, and cryopreservation of the selected cells can be realized. We are at the starting point for these great industrial possibilities.

Acknowledgments

The authors wish to express their gratitude to Dr. Katsumi Aida, Professor Emeritus, The University of Tokyo, for his extensive support and encouragement for the preparation of this monograph. The authors also acknowledge the technical assistance of Mr. K. Ohnishi, Tatoku Pearl Farm, K. MIKIMOTO & Co., LTD., for the experiment presented in Subsection 4-3. The Ministry of Agriculture, Forestry, and Fisheries of Japan supported a part of the work presented in this monograph.

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List of Figures

Fig. 1. Histological structures of the mantle of pearl oyster Pinctada fucata. A - transverse section of the marginal and the pallial zone of a mantle. A boundary between the marginal and pallial zones is indicated by a line. Outside of the mantle (o) is covered with a monolayer of epithelial cells stained in grayish blue with hematoxylin. An arrowhead indicates a periostracum gland. An arrow indicates the spot where the height of an outer epithelium changes sharply. m - marginal zone; p - pallial zone; of - outer fold; mf - middle fold; if - inner fold; pg - periostracal groove. V - ventral side; D - dorsal side; O - outside; I - inside. B - periostracal gland (arrowhead) and periostracal groove (pg). An arrow indicates periostracum. C - outer epithelial cells in the marginal zone. The outer epithelial cells that differ in the staining intensity with hematoxylin are observed. An arrowhead indicates outer epithelial cells strongly stained with hematoxylin. D - outer epithelial cells with fine acidophilic granules in the cytoplasm (arrowheads). E - a part of the pallial zone where the height of outer epithelial cells changes sharply. An arrowhead, corresponding to an arrow in A, indicates the spot where the cell height starts to change. Mayer's hematoxylin and eosin stain. Scale bars - 500 μm (A), 100 μm (B and C), 50 μm (D and E), respectively.

Fig. 2. A - presence of mucous cells (arrows) and cells with large acidophilic granules (arrowheads) in the outer epithelium. Cells with large acidophilic granules are also recognized beneath the outer epithelium. Giemsa stain. Scale bar - 50 μm. B - preparation of mantle fragments for pearl production. A ventral part of a mantle (v) is excised from a pearl oyster to prepare mantle fragments to be implanted. An inner side of the mantle is shown. After removal of the marginal zone (m), the remaining pallial zone (p) is cut into small pieces. Dark brown pigmentation is observable on the outer surface of the pallial zone (an arrow). B is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 134 pp., Fig. 5.9, © 1995, Shokabo Publishing.

Fig. 3. Histological changes in mantle allografts and pearl-sac epithelia. A - hemocyte layers (arrowheads) encapsulating the implanted bead and surrounding testis (t) at 2 days after implantation (day 2). The bead was originally at b but removed for tissue sectioning. B - a mantle allograft (a) and surrounding testis (t) on day 2. Arrowheads indicate epithelial cells flattening at the periphery of the graft. C - a mantle allograft (a) on day 4. An arrowhead indicates flattening epithelial cells. D - a pearl-sac epithelium (arrowheads) emigrating from the allograft on day 4. E - a pearl-sac epithelium (arrowheads) on day 14. An arrow indicates acidophilic granules beneath the epithelium. F - mucous cells (m) and cells with acidophilic granules (arrows) in a pearl-sac epithelium (arrowheads) on day 21. b - the place in which the bead was located; o - ovary; t - testis. Mayer's hematoxylin and eosin stain. Scale bars - 160 μm (B, C), 80 μm (A, D), 40 μm (E, F), respectively. B is reprinted with permission from Fish. Sci., 61, Awaji and Suzuki, The pattern of cell proliferation during pearl sac formation in the pearl oyster, 747–751, Fig. 1a, © 1995, The Japanese Society of Fisheries Science.

Fig. 4. DNA synthesis in a mantle tissue from an unoperated pearl oyster. BrdU-labeled nuclei were visualized with anti-BrdU monoclonal antibody and FITC-conjugated second antibody. Note the absence of BrdU labeling in the outer epithelium (o) in contrast to active labeling (arrowheads) in the inner epithelium (i). Scale bar - 200 μm. Reprinted with permission from Fish. Sci., 61, Awaji and Suzuki, The pattern of cell proliferation during pearl-sac formation in the pearl oyster, 747–751, Fig. 2a, © 1995, The Japanese Society of Fisheries Science.

Fig. 5. DNA synthesis in allografts and pearl sacs. BrdU-labeled nuclei (colored brown) were visualized with anti-BrdU monoclonal antibody and horseradish peroxidase-conjugated second antibody. Black arrowheads indicate BrdU labeling in the epithelial cells. A - a mantle allograft (a) on day 4 (an adjacent section of Fig. 3C). Inset, enlargement of the epithelium shown by a right black arrowhead. Note active labeling of the nuclei. A white arrowhead indicates absence of BrdU labeling in the central part of the allograft. B - labeling of leading squamous epithelial cells on day 4. C, D - labeling of pearl-sac epithelial cells on day 14 (C) and day 21(D). b - the place in which the bead was located; o - ovary; t - testis. Scale bars - 160 μm (A), 80 μm (B, D), 40 μm (C), respectively.

Fig. 6. Observations of pearl formation with a cover slip method. A - outgrowth of epithelial-like cells (e) on day 1. m - mantle fragment. Phase contrast. B - emigrating epithelial-like cells in flattened appearance. Phase contrast. C and D - calcified crystals appeared on the glass surface on day 11 observed with a phase contrast (C) and a polarizing microscope (D). The crystals show birefringence under polarizing microscope. Arrowheads indicate identical crystals. Scale bars - 50 μm (A), 20 μm (B), 200 μm (C, D). B is reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Fig. 37, © 1968, Fisheries Research Agency, Japan.

Fig. 7. Cover slips completely covered with nacre. Operated in June and sampled after 2 months in August. Scale bar, 2 mm.

Fig. 8. Processes of pearl-sac formation in the cover slip method. A - just after the implantation. B - the outer epithelial cells emigrate along the cover slips and proliferate. C - formation of a pearl sac. D - deposition of pearl layers around the cover slips. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Fig. 1, © 1968, Fisheries Research Agency, Japan.

Fig. 9. Decalcified pearls and adjacent pearl-sac epithelia. A–C - decalcified nacreous (A), prismatic (B), and organic (C) pearls and adjacent pearl-sac epithelia. Arrowheads in A - cells with large acidophilic granules; arrowheads in C - epithelial cells with acidophilic granules in the cytoplasm. n - nacreous layer; p - prismatic layer; o - organic layer; e - pearl-sac epithelium; m - mucous cell. D - a decalcified nacreous pearl and surrounding tissue. b - the space in which the bead was located. m - shell matrix of the implanted bead; o - organic matrix; p - prismatic layer; n - nacreous layer; e - pearl-sac epithelium; t - testis; d - digestive diverticula. Arrowhead - a transition layer between prismatic and nacreous layers. Scale bars - 20 μm (A), 150 μm (B), 250 μm (C) and 1 mm (D), respectively. D is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 161pp., Fig. 6.7, © 1995, Shokabo Publishing.

Fig. 10. Fragment culture of pearl oyster mantle with the medium shown in Table 1. A - active outgrowth of granular and agranular hemocytes on day 1. m - mantle fragment. B - outgrowth of epithelial-like cells from the mantle fragment on day 4. Granular and agranular hemocytes are observable among epithelial-like cells. Phase contrast. Scale bars - 200 μm (A) and 100 μm (B), respectively. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Fig. 58, 60, © 1968, Fisheries Research Agency, Japan.

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Fig. 11. Fragment culture of pearl oyster mantle with the TC199-based medium shown in Table 2. Outgrowth of granular and agranular hemocytes, epithelial-like cells and muscle cells. Phase contrast. Scale bar - 100 μm. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 18, Machii, Organ culture of mantle tissue of the pearl oyster Pinctada fucata (Gould), 2111–2117, Fig. 2, © 1974, Fisheries Research Agency, Japan.

Fig. 12. Deposition of dark brown secretions on the cultured mantle fragments. A - fragment culture of pearl oyster mantle in glass flasks. Some of the mantle fragments in the culture flasks are colored dark brown by the secretions (arrowheads). B - a cross section of the fully colored fragment. The fragment (m) is entirely covered with dark brown membrane-like structures (arrowhead). Delafield's hematoxylin and eosin stain. Scale bars - 10 mm (A) and 1 mm (B), respectively. A is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 179 pp., Fig. 6.15, © 1995, Shokabo Publishing.

Fig. 13. Fragment culture of pearl oyster mantle with the hemolymph-based medium. A - active outgrowth of mantle cells from a fragment on day 49. B, C - active outgrowth of round epithelial-like cells on day 7 (B) and day 8 (C). D, E - outgrown epithelial-like cells attaching to the bottom of culture vessels on day 11 (D) and day 19 (E). F - round epithelial-like cells on day 26 possessing nuclei with a prominent nucleolus (arrowhead). m - mantle fragment. Scale bars - 1 mm (A), 400 μm (B, C, E), 200 μm (D) and 100 μm (F), respectively. D is reprinted with permission from Shinju Monogatari, Popular Science, 124, Machii, 161 pp., Fig. 6.14 right, © 1995, Shokabo Publishing. E is reprinted with permission from Protein, Nucleic acid and Enzyme, 34, Machii, Tissue culture of shell, 193–196, Fig. 3, © 1989, Kyoritsu Shuppan.

Fig. 14. Fragment culture of pearl oyster mantle with the hemolymph-based medium. A–C - epithelial-like cells maintained in culture conditions for 37 days (A), 49 days (B) and 80 days (C) attaching to the bottom of culture vessels. D - membranous structures with some minute precipitates (arrowheads) observed under an outgrowth zone of the culture on day 112. The minute precipitates suggest initiation of crystal growth. m - mantle fragment. Scale bars - 400 μm (B, D) and 200 μm (A, C), respectively.

Fig. 15. Fragment culture of pearl oyster mantle with MMBSS. A - active outgrowth of mantle cells on day 1. B, C - continued outgrowth of mantle cells on day 6 (B) and day 14 (C). m - mantle fragment. Scale bars - 1 mm (A) and 200 μm (B, C), respectively.

Fig. 16. Round crystals formed on the dark brown secretions of a mantle fragment cultured for three months with the TC199-based medium. Arrows indicate identical crystals observed with three different methods. A - bright field microscopy of the crystals formed along grooves of the folds on the outer surface of the fragment. Arrowheads - dark brown precipitates deposited along the grooves. B - polarizing microscopy of the crystals. Distinct birefringence was demonstrated. C - X-ray absorption demonstrated by microradiogram preparations. Scale bars - 200 μm. B and C are reprinted with permission from Protein, Nucleic acid and Enzyme, 34, Machii, Tissue culture of shell, 193–196, Fig. 5.6, © 1989, Kyoritsu Shuppan.

Fig. 17. Trefoil-like crystals formed on the bottom of a culture vessel. Mantle fragments were cultured for 1 month with the TC199-based medium. Phase contrast (A) and polarizing microscopy (B) of the identical crystals. Scale bars - 100 μm.

Fig. 18. Shell matrix-like membranous structures formed on the bottom of culture vessels. Mantle fragments were cultured for 1 (A, C, D) or 2 months (B) with Pf35 medium with slight modifications. Arrowheads indicate thin membranous structures formed on the bottom at peripheries of the mantle fragment. Overlapping of the structures is discernible in C and D. An arrow in A indicates dark brown secretions of the fragment. Scale bars - 200 μm (A–C) and 100 μm (D), respectively.

Fig. 19. Polygonal frame-like structures observed under the outgrowth zone. Mantle fragments were cultured for 2 months with the TC199-based medium. A - frame-like structures (arrowheads) on the bottom of a culture vessel with covering cell sheets. B - frame-like structures after removal of the covering cells. Phase contrast. Scale bars - 200 μm (A) and 100 μm (B), respectively. B is reprinted with permission from Biomineralization (BIOM2001): Formation, Diversity, Evolution and Application. Machii, Pearl formation studies in vitro, 133–136, Fig. 4 left, © 2003, Tokai University Press.

Fig. 20. DNA synthesis in the outer epithelium of a cultured mantle fragment. DNA synthesis was detected in the squamous outer epithelial cells emigrating from the edge (arrowheads) but not in the columnar outer epithelial cells in the central part of the fragment (arrows). A - Mayer's hematoxylin and eosin stain. B and C - immunohistochemical detection of BrdU-labeled nuclei in an adjacent section of A with anti-BrdU monoclonal antibody and FITC- conjugated second antibody. B - phase contrast microscopy. C - fluorescent microscopy. Scale bar - 200 μm.

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Fig. 21. Procedures of excision and sterilization of a pearl oyster mantle. A - an opened pearl oyster (left) and an oyster removed of a visceral mass (right). B - disinfection of the inner surface of the mantle. C - the pallial zone (hatched) to be excised from a mantle. a - adductor muscle; m - mantle; s - shell. D - washing and mucous removal of mantle strips in MMBSS. Reprinted with permission from Invertebrate Cell Culture, Awaji, Primary culture techniques for the outer epithelial cells of pearl oyster mantle, 239–244, Fig. 1, © 1997, Science Publishers.

Fig. 22. Separation of outer epithelium from a mantle fragment digested with dispase. A - a mantle fragment observed from the outside surface showing yellowish color of the epithelium. Dark brown pigmentation of a pallial zone (an arrow) is also observable. B - a lateral view of the digested fragment. The outer epithelium (arrowheads) is recognized by its coloration. Note difference in the color of the epithelium. C - outer epithelia peeled off from the mantle fragment in the form of cell sheets. The epithelial sheets have different colors. D - aggregates of small cell sheets having different colors. E - the separated outer epithelium observed with phase contrast microscopy. The epithelial cells possess a nucleus with a prominent nucleolus. F - bright field observation of the epithelium shown in E. Dark brown pigmentation is observable (arrowheads) in some populations of the epithelial cells. Scale bars - 1 mm (A-D), 100 μm (E, F). C is reprinted with permission from Invertebrate Cell Culture, Awaji, Primary culture techniques for the outer epithelial cells of pearl oyster mantle, 239–244, Fig. 2B, © 1997, Science Publishers.

Fig. 23. Histological structures of the separated outer epithelia. A - binocular observations showing the folds of the epithelium. B - cross section of the epithelium showing a monolayer structure. a - apical surface with microvilli; b - basal surface with some connective tissue cells. C - transmission electron microscopy of the epithelium. Intercellular attachment between the epithelial cells (arrowheads) remained intact after the dispase digestion. m - mitochondria; n - nucleus; rER - rough endoplasmic reticulum. Scale bars - 500 μm (A), 50 μm (B) and 3 μm, respectively. B is reprinted with permission from Invertebrate Cell Culture, Awaji, Primary culture techniques for the outer epithelial cells of pearl oyster mantle, 239–244, Fig. 2C, © 1997, Science Publishers.

Fig. 24. Scanning electron microscopy of the separated outer epithelium. A - a piece of separated epithelium showing a monolayer structure. On the apical side (a), the top of each epithelial cell is discernible as a cupola. b - basal side. B - well-developed microvilli on the apical surface of the separated epithelium. Arrowheads - cells with rod-like structures.

Fig. 25. Dissociation of the outer epithelial cell clumps by pronase at various concentrations. Digestion of the epithelial clumps at 10 mg/ml was suitable for the dissociation. A - 1 mg/ml; B - 5 mg/ml; C - 10 mg/ml. Scale bars - 100 μm.

Fig. 26. The epithelial clumps cultured on a hemocyte sheet. A - 1 h after the inoculation. The epithelial clumps (arrows) have started to adhere to the hemocyte sheet (h). B and C - 1 day after the inoculation. In B, the floating cells liberated from the epithelial clumps and hemocyte sheets are covering the bottom of the well. Arrow - an epithelial clump changing its shape. In C, the epithelial cells have started to migrate (arrows) from the clump (c). Scale bars - 200 μm (A) and 100 μm (B, C), respectively. With kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 2A–C.

Fig. 27. Monolayer epithelial cell sheets formed on the hemocyte layers. The outer epithelial cells (e) adhered to the hemocyte layers (h) at the basal side, and possessed microvilli (arrowheads) on the apical side - A, a relatively thick monolayer of the epithelial cells at the central part of the cell sheet. B - the peripheral part of the epithelial cell sheet. The epithelial cells became flattened, and acellular spaces often occurred between hemocyte layers and the epithelial sheet. C - an extremely flattened epithelial cells at the margin of the cell sheet. p - bottom of the plastic well. Scale bars - 5 μm.

Fig. 28. Ultrastructure of the epithelial cell sheet. A - central part of the epithelial cell sheet. B - cell-to-cell junction observed in the epithelial cell sheet. C - cell-to-cell junction of the squamous epithelial cells. e - the epithelial cell sheet; h - hemocyte layer; m - microvilli; s - septate junction; d - desmosome. Scale bars - 1 μm (A) and 0.5 μm (B, C), respectively. B and C are reprinted with kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 3B, D.

Fig. 29. A - the epithelial cell clumps inoculated into a plastic well without hemocytes. B - the epithelial clumps cultured on the fixed hemocyte sheet on day 7. The epithelial cell clumps (e) adhered to the fixed hemocytes (h) but did not form a monolayer sheet. Spherical cells were liberated from the clumps. Scale bars - 100 μm. B is reprinted with kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 2D.

Fig. 30. Detection of DNA synthesis in epithelial cell sheet. The cultures on day 6 were labeled with BrdU for 24 h and immunohistochemically stained with anti-BrdU antibody to visualize DNA synthesis in the cells. A part of the immunoreactive nuclei is indicated by black arrowheads. White arrowheads indicate nuclei without positive reactions. A–C - the epithelial cell sheet with different sizes and labeling frequency. D - the epithelial cell sheet without positive reactions. Scale bars - 100 μm. A and B are reprinted with kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Fig. 4.

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Fig. 31. Trial of pearl production with cultured mantle cells. A - cells outgrowing from the mantle fragment on day 1. B - cell suspension used for the injection. Cells outgrown from the mantle fragments (shown in A) were harvested by digestion of cell sheets with pronase. C - one of the 13 beads retrieved from the oysters was completely covered with nacreous layers (arrowhead), but the others were without pearl layers of any kind. Scale bars - 100 μm.

Fig. 32. Zoosporic fungi appearing in a cell culture from veliger larvae of P. fucata. Cell clusters resembling animal cells are all fungus cells. The clusters attach to the bottom firmly by many pseudopodia-like extensions observable around the cell clusters (arrowheads). Scale bar - 100 μm. Reprinted with permission from Commun. Appl. Cell Biol., 15, Machii, In vitro studies of pearl formation, 1–10, Fig. 7, © 1998, Japan Academy of Applied Cell Biology.

Table 1. Tissue culture medium used for early studies on mantle fragment culture of pearl oyster Pinctada fucata. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 13, Machii, Histological studies on the pearl-sac formation, 1489–1539, Table 2, © 1968, Fisheries Research Agency, Japan.

Table 2. Tissue culture medium based on TC199 used for mantle fragment culture of pearl oyster Pinctada fucata. Reprinted with permission from Bull. Natl. Pearl Res. Lab., 18, Machii, Organ culture of mantle tissue of the pearl oyster Pinctada fucata (Gould), 2111–2117, Table 1, © 1974, Fisheries Research Agency, Japan.

Table 3. Balanced salt solution (MMBSS) and calcium/magnesium-free phosphate buffer (MMCMF) for marine mollusks. Used with permission of CRC Press, from Some marine invertebrates tissue culture, Machii and Wada, In: J Mitsuhashi (ed). Invertebrate Cell System Applications, Vol. II, 1989; permission conveyed through Copyright Clearance Center, Inc.

Table 4. Tissue culture medium Pf35 for pearl oyster Pinctada fucata. Used with permission of CRC Press, from Some marine invertebrates tissue culture, Machii and Wada, In: J Mitsuhashi (ed). Invertebrate Cell System Applications, Vol. II, 1989; permission conveyed through Copyright Clearance Center, Inc.

Table 5. Effects of the hemocyte layers on the adhesion of the mantle epithelial cells. With kind permission from Springer Science + Business Media: In Vitro Cell. Dev. Biol., Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes, 34, 1998, 486–491, Awaji and Suzuki, Table 1.

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