Summary
In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 µM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.
Similar content being viewed by others
References
Awaji, M. Separation of outer epithelial cells from pearl oyster mantle by Dispase digestion. In: Fraser, M. J., Jr., ed. Proceedings of the Eighth International Conference on Invertebrate and Fish Tissue Culture. Columbia, MD: Tissue Culture Association; 1991:43–46.
Awaji, M.; Suzuki, T. The pattern of cell proliferation during pearl sac formation in the pearl oyster. Fisheries Sci. 61:747–751; 1995.
Bennett, N. T.; Schultz, G. S. Growth factors and wound healing: Part II. Role in normal and chronic wound healing. Am. J. Surg. 166:74–81; 1993.
Bohn, H. Growth-promoting effect of haemocytes on insect epidermis in vitro. J. Insect Physiol. 21:1283–1293; 1975.
Bohn, H. Enzymatic and immunological characterization of the conditioning factor for epidermal outgrowth in the cockroach Leucophaea maderae. J. Insect Physiol. 23:1063–1073; 1977.
DeVore, D. P.; Engebretson, G. H.; Schachtele, C. F. Identification of collagen from byssus threads produced by the sea mussel, Mytilus edulis. Comp. Biochem. Physiol. 77B:529–531; 1984.
Gailit, J.; Clark, R. A. F. Wound repair in the context of extracellular matrix. Curr. Opin. Cell Biol. 6:717–725; 1994.
Kimura, S.; Karasawa, K. Squid cartilage collagen: isolation of type I collagen rich in carbohydrate. Comp. Biochem. Physiol. 81B:361–365; 1985.
Kimura, S.; Tanaka, H. Characterization of top shell muscle collagen comprising three identical α1 chains. Bull. Jpn. Soc. Sci. Fish. 49:229–232; 1983.
Kimura, S.; Takema, Y.; Kubota, M. Octopus skin collagen: isolation and characterization of collagen comprising two distinct α chains. J. Biol. Chem. 256:13230–13234; 1981.
Lebel, J.-M.; Giard, W.; Favrel, P., et al. Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc, Haliotis tuberculata. Biol. Cell. 86:67–72; 1996.
Machii, A.; Wada, K. T. Some marine invertebrates tissue culture. In: Mitsuhashi, J., ed. Invertebrate cell system applications. Vol. 2. Boca Raton, FL: CRC Press; 1989:225–233.
Mizuta, S.; Yoshinaka, R.; Sato, M., et al. Subunit composition of two distinct types of collagen in the muscle of the squid Todarodes pacificus. Fisheries Sci. 60:597–602; 1994.
Odintsova, N. A.; Nesterov, A. M.; Korchagina, D. A. A growth factor from tissues of the mussel Mytilus edulis. Comp. Biochem. Physiol. 105A:667–671; 1993.
Sanford, K. K.; Earle, W. R.; Evans, V. J., et al. The measurement of proliferation in tissue culture by enumeration of cell nuclei. J. Natl. Cancer Inst. 11:773–795; 1951.
Suzuki, T.; Funakoshi, S. Isolation of a fibronectin-like molecule from a marine bivalve, Pinctada funcata, and its secretion by amebocytes. Zool. Sci. 9:541–550; 1992.
Suzuki, T.; Yoshinaka, R.; Mizuta, S., et al. Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oyster Pinctada funcata. Cell Tissue Res. 266:75–82; 1991.
Tsujii, T. Studies on the mechanism of shell- and pearl-formation in Mollusca. J. Fac. Fish. Prefect. Univ. Mie 5:1–70; 1960.
Wada, K. Studies on the mineralization of the calcified tissue in molluscs-XIII. Histological and histochemical studies of mucous cells on the inner and shell surfaces of mantle of some bivalve and gastropod molluscs. Bull. Natl. Pearl Res. Lab. 11:1283–1297; 1966.
Wada, K.; Shintani, H. The behaviour of outer mantle epithelial cells implanted into the mantle connective tissue of the freshwater mussel, Hyriopsis schlegeli at an early stage of pearl-sac formation. Venus Jpn. J. Malacol. 54:133–142; 1995.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Awaji, M., Suzuki, T. Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes. In Vitro Cell.Dev.Biol.-Animal 34, 486–491 (1998). https://doi.org/10.1007/s11626-998-0083-0
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11626-998-0083-0