Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex trunculus digestive cells

Abstract

Background: Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food.

Results: The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells.

Conclusion: The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids.

Figure 1

Hexaplex trunculus. (A), Hexaplex trunculus, the shell has been removed in the posterieur side but the mantle is intact, marine snail sagittale section showing the hepatopancreas in the posterieur side. (B), Hexaplex trunculus hepatopancreas showing the epithelium of the digestive diverticula displaying the small lumen and the interstitial tissue. Sections were stained with eosine and hematoxyline.

Figure 2

Immunoblot. Immunoblot analysis of 5 μg mSDPLA₂ (lane 1); 50 μg mSDPLA₂ (lane 2); 15 μg mSDPLA₂ (lane 3); PPLA₂ (lane 4). Using mSDPLA₂ serum at 1: 1000 dilution.

Figure 3

Hexaplex trunculus Cryostat tissue sections (× 400) through digestive diverticula. Frozen 4 μm sections from H. trunculus hepatopancreas were used for control experiments (without pAbs anti-mSDPLA₂) and stained with eosine and hematoxyline for genral morphology. No labeling was observed without pAbs anti-mSDPLA₂. (A and B) Overall view of a control section of the digestive diverticula at low magnification (100×). (C and D) Enlarged view of the digestive diverticula sectioned longitudinally (400×).

Figure 4

Immunalabelling of the digestive gland. (A and B) Overall view of a sections of the digestive diverticula after incubation with the solution containing pAbs anti-mSDPLA₂ (1:100) and then hybridized with appropriate secondary biotinylated- antibodies (1:200) counter-stained with eosine and hematoxylin. (C and D) The sectioon of digestive diverticula showing the occurrence of mSDPLA₂ in the lumen of digestive cells, mSDPLA₂ localize in the around cells. (E and F) enlarged view of the digestive cells (400×).

Figure 5

Immunofluorescence localization of mSDPLA₂ in a 4-μm cryostat section. (A) One section was first stained with Di Aminido Phenyl Indol (DAPI) for general morphology. (B) Followed by pAbs anti-mSDPLA₂ (1:100) revealed by fluorochrome (chromeo) conjugated with the secondary antibody in PBS (1:200). (C) represents the superposition of two images (A and B), showing labelling of digestive cells intracellular granules.

Figure 6

Electron micrographs of the digestive cells. (A, B,) the apical region of the cell contains a bruch border of microvilli and numerous clumps of α-glucogen granules. (C) digestive adjacent cells. Note some microtubules (arrow) and a ciluim root (arrowhead). (D) The cytoplasm of digestive cells contain granules similar to hemozoin, vesicles with protein content and lipid droplets, indicating the possible function in digestion, absorption, and storage of lipids. (E) The pinocytotic vesicles fuse together to form heterogeneous phagosomes known as heterophagosomes. (F) A large residual body is about to extruded into lumen at the end of the process. (G) The vacuola become bigger and distributed through the whole cell.