BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

John De La Cuesta

PROJECT TITLE:

Formation of Biomineralized granules in the Digestive Gland of Littorina littorea

Species Identification

• Kingdom: Animalia
  
  
• Phylum: Mollusca
  
  
• Class: Gastropoda
  
  
• Order: Prosobranchia
  
  
• Family: Littorinacae
  
  
• Genus: Littorina
  
  
• Species: littorea

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Abstract:

Biomineralized granules are found in the digestive gland of Littorina littorea. They exist primarily in the basophil cell. Transmission electron microscopy of these cell types showed granules in differing stages of development. Some mitochondria contained electron dense formations which points to the possibility of intramitochondrial formation of biomineralized granules.

Key Words:

Littorina littorea, biomineralization, digestive gland, intracellular granules, TEM.

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INTRODUCTION

The digestive gland of Littorina littorea has been the focus of many recent studies on toxicology (Nott and Langston, 1989; Mason, Simkiss and Ryan, 1984). The primary cell type studied has been the basophil cell which contains calcium pyrophosphate granules. It is these granules that are the site of assimilation of certain heavy metals (Nott and Langston, 1989). Other functions have been proposed as well and are summarized by Mason and Nott (1981).


Currently, there are no studies that have explained the formation of granules in prosobranch mollusks. Studies of granule formation in other invertebrates have shown the cytoplasmic vesicle and cisternae of the endoplasmic reticulum to be the initial sites of formation (Simkiss, 1976). Calcium granules have also been documented as forming in the mitochondria of calciferous gland cells in earthworms (Crang, Holden and Hitt, 1968). Calcium phosphate granules have also been shown to form in hepatopancreas mitochondria of the blue crab Callinectes sapidus (Chen, Greenawalt and Lehninger, 1974).

Although intramitochondrial formation of calcium granules is the exception and not the rule (Simkiss, 1976), results of this study indicate that this mechanism may occur in Littorina littorea.

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MATERIALS AND METHODS

Whole animals (Littorina littorea) were removed from their shells. Digestive glands were dissected out and cut into 1 cubic mm pieces. The tissue was fixed in 2% gluteraldehyde for one hour. The tissue was then fixed in 1% osmium tetroxide for one hour. The fixatives were buffered with 0.2M sodium cacodylate at pH 7.6. The tissue was dehydrated in graded alcohols and propylene oxide then embedded in Spurr low viscosity resin (Spurr, 1969). Thick and ultrathin sections were cut on a Sorvall MT2B Ultramicrotome. Ultrathin sections were double stained with uranyl acetate and lead citrate (Reynolds, 1963) and examined in a JEOL 1200 EX II transmission electron microscope. The accelerating voltage was set at 80 kV for all micrographs with film sensitivity of 17.

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RESULTS

Figure 1.



TEM image of digestive cell. (v) vacuole; (n) nucleus. X 4000. Bar=2 microns.

Figure 2.



TEM image of digestive cell. (V) vacuole. Bar=l microns.

Figure 3.



TEM image of basophil cell. (ps) protein secretion. X6000. Bar=l microns.

Figure 4.



TEM image of basophil cell . (ps) protein secretion; (er) rough endoplasmic reticulum. X25K. Bar=200 nm.

Figure 5.



TEM image of basophil cell. (ps) protein secretion. (m) mitochondria; (g) golgi apparatus; arrow indicates cell membrane. X15K. Bar=500 nm.

Figure 6.



TEM image of basophil cell. (gr) granule. X5000 Bar=l micron.

Figure 7.



TEM image of granule. (gr) granule; arrow indicates cell membrane. X15K. Bar=500 nm

Figure 8.



TEM image of granule. (gr) granule; arrow indicates vesicular boundary, X80K. Bar=100 nm.

Figure 9.



TEM image of granule formation. (gr) granule; (nu) nucleus; arrows indicate forming granule. X6000. Bar=l micron.

Figure 10.



TEM image of electron dense bodies. Arrows indicate electron opaque formations. X25K. Bar=200 nm.

Figure 11.



TEM image of electron dense bodies within mitochondrion. (c) cristae; arrow indicates membrane. X60K. Bar=100nm.

Figure 12.



TEM image of electron dense bodies within mitochondrion. (c) cristae; arrow indicates membrane. X100K Bar=50nm.

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DISCUSSION

The digestive gland of Littorina littorea consists of two primary cell types: basophil cells and digestive cells. Digestive cells are characterized by large vacuoles (figures 1 and 2) which are presumably the site of intracellular digestion (Mason, Simkiss and Ryan, 1984). Basophil cells (figure 3) are less numerous than digestive cells but are easily recognized due to their electron dense protein secretions (Mason, Simkiss and Ryan, 1984). These cells are characterized by protein secretions (figure 4), well defined rough endoplasmic reticulum, Golgi apparatus, mitochondria (figure 5), and numerous calcium granules (figure 6).

Many calcium granules were found in basophil cells (figures 6.7 and 8). They are bound by membranes and tend to differ in size from 1 to 5 micrometers in diameter. This is probably due to the fact that they are spherical in shape (Nott and Langston 1989) and that section have been cut at different levels of the sphere, The granules consist mostly of insoluble calcium phosphate salts (Nott and Langston, 1989; Mason and Nott, 198l) which do not section well with a glass knife. Many of the granules are simply plucked out during sectioning which accounts for the numerous holes in the sections. Figure 8 shows an electron dense area in the middle of the granule while outer portions are more penetrable by the beam. This granule appears to be a fully formed B type III due to its morphology (Simkiss, 1976). The membrane surrounding the granule suggests that it may be formed within a vesicle (Simkiss, 1976), or perhaps a membrane bound organelle (Mason and Simkiss, 1982).

Granules just beginning to form (figures 9 and 10) tend to be quite electron dense (Simkiss, 1976) and may show concentric ring-like formation. Figure 9 shows small granules beginning to form inside a vesicle which appears to be a degenerated mitochondrion. It is therefore possible that intramitochondrial formation of calcium granules may occur in Littorina littorea (figures 11 and 12). Electron dense material is forming inside the mitochondria. It is inferred that these are primary calcium granule formations due to their electron density. Organic material may be associated with these intramitochondrial formations as well (Simkiss, 1976). This may account for their morphology. Whether or not these formations are due to fixation artifact is unknown.

Certainly, this ultrastructural study cannot conclude that mitochondria are the site of calcium granule formation. Mitochondria do however posses the transmembrane cation pumps necessary to accomplish biomineralization (Mason and Simkiss, 1982). Further studies on intramitochondrial formations should include energy dispersive X-ray microanalysis and pulse chase autoradiography to determine elemental composition, routes and temporal data in relation to possible biomineralization in these organelles.

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CITATIONS

Chen, C., Greenawalt, J.W., and Lenninger, A.L.. (1974). Biochemical and ultrastructural aspects of Ca transport by mitochondria of the hepatopancreas of the blue crab Calinectes sapidus. The Journal of Cell Biology, Vol.61, 301-315.

Crang, R.E., Holsen, R.C., and Hitt, J.B. (1968). Calcite production in mitochondria of earthworm calciferous glands. Bioscience. Vol.18, 299-301.

Mason, A.Z., and Nott, J.A. (1981). The role of intracellular biomineralized granules in the regulation and detoxification of metals in gastropods with special reference to the marine prosobranch Littorina littorea. Aquatic Toxicology. Vol.1, 239-256.

Mason, A.Z., and Simkiss, K. (1982). Sites of mineral deposition in metal accumulating cells. Experimental Cell Research. Vol.139, 383-391.

Mason, A.Z., Simkiss, K., and Ryan, K.P. (1984). The ultrastructural localization of metals in specimens of Littorina littorea collected from clean and polluted sites. J. Mar. Biol, Ass. U.K, Vol,64, 699-720.

Nott, J.A., and Langston, W.J. (1989). Cadmium and the phosphate granules in Littorina littorea. Mar. Biol. Ass. U.K. Vol, 69, 219-227.

Reynolds , F S . ( 1963). The use of lead citrate at high pH as anelectron-opaque stain in electron microscopy, Journal of Cell Biology. Vol.17, 208-212.

Simkiss, K. (1976). Intracellular and extracellular routes in biomineralization. Symposium of the Society for Experimental Biology. Vol.30, 423-444.

Spurr, A.R., (1969). Journal of Ultrastructural Research. Vol.26, 31.