Okajimas Folia Anat. Jpn., 72(5): 277-284, December, 1995 Immunocytochemical Evidence for the Presence of Prolactin in the Plerocercoid of Ligula Intestinalis (Cestoda: Pseudophyllidea) By Bo LIU, Hidekazu WAKURI and Ken-ichiro MUTOH Department of Animal Anatomy, Changchun University of Agriculture and Animal Sciences, Xian Road 175, Changchun, Jilin 130062, P.R. China Department of Veterinary Anatomy, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan -Received for Publication, November 9,1995- Key Words: Prolactin, Plerocercoid, Cestode, Nervous system, Immunocytochemistry Summary: Immunoreactivity to prolactin in the nervous system of the plerocercoid of Ligula intestinalis was demonstrated by immunocytochemical method. Numerous PRL immunoreactive perikarya with long varicose fibres were observed in the peripheral nervous system in the worm, mainly in the transversal muscle layer and medullary parenchyma of the midbody. A few fibres were found in the main nerve cords of the central nervous system. PRL positive neurons sent their processes to associate with the main nerve cords. The immunostaining terminals appeared in the subtegument region in the lateral border of the plerocercoid. The result indicates that PRL immunoreactivity is well-developed in the plerocercoid of the cestode. The significance of the localization of prolactin in the worm is discussed. Recent 'years a interest in the neuropeptides in immunoreactivity in the Hymenolepis nana using the platyhelminthes is growing. There are no true human prolactin antibody has been reported endocrine glands and a circulatory system in these (Kumazawa & Moriki, 1986). organisms, and so the neurosecretory (peptidergic) The aim of the present study was to explore the component of the nervous system probably serves an prolactin immunocytochemicalevidence for the pre- important role in the flatworm. They may be an sence and distribution in the plerocercoid of Ligula exploitable target for chemotherapeutic evaluation. intestinalis. A number of description of mammalian and inverte- brate neuropeptides immunoreactivity in the platy- helminthes is given by researchers (Marks et al., Materials and Methods 1993; Maule et al., 1992; Gustafsson et al., 1995; Fairweather et al. , 1990). Plerocercoid of Ligula intestinalis (Pseudophyl- Prolactin (PRL) have been shown to have multiple lidea) were obtained from fish (Crucian carp) from functions including the osmoregulation, regulation Lake Taipinhu, China. The plerocercoid were taken of metamorphosis, leuteotropic and leuteolytic from the body cavity. Worms were fixed directly in effects,proliferative actions and regulationof parental 4% paraformaldehyde after washing with physio- behavior, etc. in the mammalian and nonmammalian logical saline solution, sectioned at freezing micro- species. It was previously believed to occur exclu- tome at 30-60 itim thickness and stained with the sively in the adenohypophyseal endocrine cells, immunocytochemical method avidine-biotin com- but recent immunoassay and immunocytochemical plex (ABC), using the following procedure: sections studies have been revealed that PRL neurons are were washed in PBS and 0.2% Triton X-100, incu- localized in the hypothalamus and other nuclei of the bated with normal goat serum for 30 min, rinsed in brain in the vertebrates (Emanuele et al. , 1986; PBS and then incubated in primary antiserum — Berghman et al., 1992; Wright, 1986; Harlan et al., rabbit anti-prolactin (sheep) polyclonal antibody 1989). Indeed, PRL can be regarded as an adeno- (Chemicon international INC.) diluted 1:1000 for phypophyseal hormone as well as a neuropeptide. 48-60 h at 4°C, rinsed in PBS and incubated in There is relatively few data on prolactin in the biotinylated antibody for 1 h at room temperature. flatworm, only the demonstration of prolactin After rinsing in PBS again, the sections were incu- 277 278 Liu, B et al. bated in avidine-biotinylated horseradish peroxidase the ganglia. A few fibres were observed in the main complex (Vector laboratories, CA, U.S.A) diluted nerve cords belonging to the central nervous system 1:100 in PBS for 1 h at room temperature. Following (Fig. 5). Thin PRL immunoreactive fibres extended another rinse, the sections were processed for per- from the main nerve cords to the peripheral nervous oxidase activity in 0.02% DAB/0.005% H202 for system. In the transversal muscle layer and the 10 min at room temperature. The sections were medullary parenchyma many PRL immunoreactive washed in PBS, mounted on slides, dehydrated, neurons were showed to stretch their processes into coverslipped, and then observed under the light the main nerve cords in right angle (Fig. 6), and then microscopy. run along the lateral of the cords or cross them to the Controls included omission of primary antiserum cortical or medullary parenchyma. and substitution of primary antiserum with non- In the region of the genital anlage formation PRL immune rabbit serum or PBS. immunoreactive neurons and fibres were scattered. A few fibres passed through on the surface of the genital anlageor ended to the formation.PRL labelled Results neurons located near the genital anlage were showed to send short processesto associatewith them (Fig. 8). The distribution of PRL immunoreactive neurons The small spindle cells, at 4-611m in diameter, were extensively observed in the plerocercoid, par- were usually distributed in the dorsal or the ventral ticularly in the peripheral nervous system. The PRL areas of the main nerve cords. Positive fine granules immunostaining cell, usually in bipolar and multi- were seen in the perikarya. They were oblique in the polar, had small perikarya, 5 —121..tm in diameter, position, usually a few short positive nerve fibres with unstained large round-nucleus. Strong positive were distributed close to them in same direction granules were scattered in the cytoplasm, mainly (Fig. 7). arranged surround the nucleus as the perinuclear Large cells (14-201m in diameter), round in granules (Fig. 1). These cells with long processes shape, with two thick tube-like processes were ob- were located mainly in the transversal muscle layer served in the medullary parenchyma. A comparative and the medullary parenchyma, partially in the cor- weak immunoreactivitywas showed in the perikarya tical parenchyma in the midbody. Only a few peri- (Fig. 9). PRL immunoreactivefibres from the neurons karya with the immunoreactivity were recognized in usually extended along the processes of the large the longitudinal muscle layer and both the anterior cells and ended the coarse immunoreactive terminals part and posterior part of the worms. on the surface of these processes. PRL positive Dense accumulation of immunostaining fibres filled neurons contacting with the large cells were found in with large granular material was clearly showed in the worm (Fig. 10). the midbody, sparsely in the anterior and posterior The controls gave the negative immunoreaction. parts of the worm. Numerous long varicose fibres from the PRL perikarya extended to the main nerve cords and laterally to the border of the body. There- Discussion fore, many fibres were observed running in transversal direction and perpendicular to the longitudinal axis The present study reveals first demonstration of of the body from the longitudinal sections (Fig. 2). PRL immunoreactivity in the plerocercoid of Ligula In the cross sections positive reactive cells usually intestinalis. PRL immunoreactivity in the Hymeno- stretched fibres to the lateral border, few fibres lepis nana has been demonstrated, but only located extending to the dorsal and ventral border in the in the anterior periphery of the retracted rostellum worms. and within the rostellar tegument (Kumazawa & Positive fibres on the way of extending were showed Moriki, 1986). Negative results of PRL immuno- to send collaterals connecting each other and forming reactivity in the Schistosoma spp. , Fasciala and fibre network (Fig. 3). Some PRL fibres in the Echinostoma spp. were detected in other research medullary parenchyma passed through the main nerve (review. Fairweather & Halton, 1991). Very different cords extending to the cortical parenchyma, where result was obtained in the present study. The result they united with fibres from the local PRL neurons shows the PRL immunoreactive cells are well- to form the fibre plexa and then terminated close to developed and widely distributedin the plerocercoids. the opening of the peripheral excretory duct and the They were located chiefly in the peripheral nervous subtegument region (Fig. 4). Only a few nerve fibres system of the worm. were seen to extended toward the basal lamina of The cytomorphology of the PRL immunoreactive the tegument. cells conforms with that of the paraldehyde fuchsin PRL immunoreactive perikarya was not seen in (PAF) positive neurons described from Diphyllo- Prolactin Immunoreactivity in Plerocercoid 279 bothrium dendriticum by Gussstafsson & Wikgren PRL immunoreactive cell system in the larva of (1981).PRL immunostainingcells have many distinct cestode favors the view that the PRL may act as granules in the cytoplasm and varicose fibres. As transmitter or modulator and involve in the prolifer- flatwormhave no a circulatory system, the need for a ative and differentiative in the development of larva dispersed population of secretory neurons is great. and during the change of host from fish to bird. The present result supportsthe views that the nervous It is interesting