In vitro Uptake of Radioglucose by a Frog Lung-Fluke and Correlation with the Histochemical Identification of Glycogen

Abstract

Haematoloechus medioplexus from Rana pipiens were tested histochemically for glycogen by using the Bauer-Feulgen method. Major concentrations of glycogen occur in the oral sucker, pharynx, anterior subcuticular parenchyma, and spermatozoa. Worms maintained in vitro in glucose-saline showed no depletion of tissue glycogen after 50 hr, while worms maintained in only saline showed a partial loss of glycogen from the oral sucker, pharynx, and subcuticular parenchyma. In other experiments worms were maintained in vitro in saline containing radioglucose. After 12, 24, and 48 hr worms were sectioned and autoradiographed and compared with sections treated histochemically. The distribution of radioactivity corresponded with that of glycogen, indicating that glucose was removed from the medium and synthesized into glycogen. Grain counts were made to determine the amount of radioactivity (as C14 glycogen). The most radioactive structures were the pharynx and the anterior subcuticular musculature and parenchyma. Considerable glycogenesis also occurs in the oral sucker and deep parenchyma, but relatively little glycogen synthesis occurs in the cuticle, Mehlis' gland, vitelline follicles, digestive ceca, and ovary. Spermatozoa synthesize glycogen only shortly after their formation. Older spermatozoa in seminal receptacles were not radioactive. In this research histochemical and autoradiographic techniques were utilized in studying the uptake and fate of exogenous glucose in a frog lung-fluke, Haematoloechus medioplexus Stafford (Trematoda: Plagiorchiidae). The objectives were to obtain a more detailed understanding of the localization of glycogen and to obtain data on relative rates of glucose uptake by various tissues. Two publications have dealt with the distribution of glycogen in H. medioplexus as shown by histochemical methods. Axmann (1947) used a key with subjective values from 0 (no glycogen) to (large number of glycogen granules) to describe the distribution and appearance of glycogen in various structures. Burton (1960), in a preliminary study, largely substantiated Axmann's findings and made certain supplementary observations. MATERIALS AND METHODS H. medioplexus was obtained from lungs of Rana pipiens purchased from commercial sources. Received for publication 25 July 1962. * This research was supported by grants from the Atomic Energy Commission [AT(11-1)-1016] and the National Science Foundation (G17657). Frogs were pithed and the lungs quickly removed and placed in Ringer's amphibian saline solution. Lungs were opened and, if infested, worms were removed and washed in Ringer's solution prior to further processing. Worms to be sectioned were slightly flattened under a cover glass and( fixed in absolute alcoholformalin (9:1) for 12 to 24 hr at 4 to 5 C. The Bauer-Feulgen method was used to determine the distribution of glycogen within tissues. Worms were sectioned at 10 to 12 ,u and serially mounted on slides. Sections were oxidized in 4% chromic acid for 1 hr and exposed to Feulgen reagent for 15 min; no counterstain was used. Control sections were treated with a 0.1% solution of malt diastase in buffered neutral saline for 1 hr at 37 C. The solution in which worms were maintained in vitro was made up by adding 20,000 units of Penicillin G and 1 img of streptomycin per 10 ml of Ringer's solution. In some experiments glucose was added to make 1 and 2% solutions. Worms to be isolated were washed in several changes of antibiotic saline after being selected by examination through a dissecting microscope. For specimens to be isolated in vitro and then studied histochemically, the following procedure was employed: after washing, no more than 5 worms were placed in 10 ml of the appropriate medium contained in a stender dish, the dish was then covered and kept at room temperature (22 to 25 C). In the autoradiographic studies two identical