Tissue and Cell 39 (2007) 247–256
TEM immunocytochemistry of a 48 kDa MW organic matrix
phosphoprotein produced in the mantle epithelial cells
of the Eastern oyster (Crassostrea virginica)
, M.B. Johnstone
, A.S. Mount
, H. Silverman
, A.P. Wheeler
Department of Biological Sciences, Clemson University, Clemson, SC 29634, United States
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, United States
Received 12 March 2007; received in revised form 14 March 2007; accepted 15 March 2007
Immunohistochemical TEM of Eastern oyster (Crassostrea virginica) mantle epithelial cells using a polyclonal antibody to a gel puriﬁed
48 kDa MW oyster shell phosphoprotein revealed that it is phosphorylated in the Golgi, packaged into secretory vesicles and subsequently
exocytosed across the apical membrane of specialized cells. These phosphoprotein producing cells are concentrated along the mantle side
facing the shell, in the region of the outer mantle lobe. A layer of calcium enriched immuno-reactive mucous is associated with the apical
microvilli of these cells. The 48 kDa phosphoprotein forms a component of the ﬁbrous organic matrix and appears to be involved in calcium
supply thus enabling crystal growth at the mineralization front.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Ultrastructure; Secretory cells; Mantle; Biomineralization; 48 kDa phosphoprotein; Oyster; Organic matrix
The molluscan mantle organ forms the outer integument
that encloses the body of the animal. It functions to provide
protective mucous secretions and participates in shell forma-
tion (Simkiss and Wilbur, 1989). The organ is composed of
two epithelia, a gill facing pallial epithelium and a shell fac-
ing outer mantle epithelium (OME). Conﬁned within these
two layers are connective, nervous and muscle tissues as well
as circulating immune cells (hemocytes), blood vessels and
hemocoels which comprise a portion of the open circulatory
system of the animal.
In bivalves, such as the Eastern oyster, the outer margin of
the mantle elaborates into three lobes which are involved in
the formation of new shell. Epithelial cells lining the surface
Corresponding author at: Clemson University, Department of Biological
Sciences, 132 Long Hall, Clemson, SC 29634, United States.
Tel.: +1 864 656 3597; fax: +1 864 656 0435.
E-mail address: email@example.com (A.S. Mount).
of the outer lobe, the underlying middle lobe and the inter-
secting region (between the middle and inner lobes) termed
the periostracal groove secrete a variety of proteins, lipids
and carbohydrates that contribute to the formation of the
organic matrix of calciﬁed shell (Gr
egorie, 1972; Lowenstam
and Weiner, 1989). In Crassostrea virginica, this organic
fraction accounts for less than 1% of the dry weight of
shell (Kawaguchi and Watabe, 1993; Mount, 1999; Rusenko,
The function of the complex assemblage of organic matrix
macromolecules in shell formation is poorly understood.
Only general functions have been proposed based on his-
tochemical examination of the tissue and largely in vitro
experiments from shell extracts (Addadi et al., 2006). In
the Eastern oyster, glycosaminoglycans (GAGs) have been
observed surrounding calcite crystals in situ (Kawaguchi and
Watabe, 1993) and phosphoprotein extracts have been shown
to bind calcium carbonate crystals when immobilized in a
hydrogel (Mount, 1999) and inhibit crystal growth when in
solution (Wheeler et al., 1981, 1987).
0040-8166/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.